DYRK1A inhibition suppresses STAT3/EGFR/Met signalling and sensitizes EGFR wild‐type NSCLC cells to AZD9291

DYRK1A is considered a potential cancer therapeutic target, but the role of DYRK1A in NSCLC oncogenesis and treatment requires further investigation. In our study, high DYRK1A expression was observed in tumour samples from patients with lung cancer compared with normal lung tissues, and the high levels of DYRK1A were related to a reduced survival time in patients with lung cancer. Meanwhile, the DYRK1A inhibitor harmine could suppress the proliferation of NSCLC cells compared to that of the control. As DYRK1A suppression might be effective in treating NSCLC, we next explored the possible specific molecular mechanisms that were involved. We showed that DYRK1A suppression by siRNA could suppress the levels of EGFR and Met in NSCLC cells. Furthermore, DYRK1A siRNA could inhibit the expression and nuclear translocation of STAT3. Meanwhile, harmine could also regulate the STAT3/EGFR/Met signalling pathway in human NSCLC cells. AZD9291 is effective to treat NSCLC patients with EGFR‐sensitivity mutation and T790 M resistance mutation, but the clinical efficacy in patients with wild‐type EGFR remains modest. We showed that DYRK1A repression could enhance the anti‐cancer effect of AZD9291 by inducing apoptosis and suppressing cell proliferation in EGFR wild‐type NSCLC cells. In addition, harmine could enhance the anti‐NSCLC activity of AZD9291 by modulating STAT3 pathway. Finally, harmine could enhance the anti‐cancer activity of AZD9291 in primary NSCLC cells. Collectively, targeting DYRK1A might be an attractive target for AZD9291 sensitization in EGFR wild‐type NSCLC patients.     

Identification of osimertinib (AZD9291) as a lysine specific demethylase 1 inhibitor

Osimertinib (AZD9291) is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) that has been approved for the treatment of EGFR-mutated non-small cell lung cancer (NSCLC). In this study, osimertinib was characterized as a LSD1 inhibitor for the first time with an IC₅₀ of 3.98 ± 0.3 μM and showed LSD1 inhibitory effect at cellular level. These findings provide new molecular skeleton for dual inhibitor for LSD1 and EGFR. Osimertinib could serve as a lead compound for further development for anti-NSCLC drug discovery with dual targeting.     

A natural anthraquinone derivative shikonin synergizes with AZD9291 against wtEGFR NSCLC cells through reactive oxygen species-mediated endoplasmic reticulum stress

BackgroundNSCLC is the major type of lung cancer and the survival rates of NSCLC patients remain low. AZD9291 is a third-generation EGFR-TKI and approved to treat NSCLC patients harboring EGFR T790M mutation and common targetable activating EGFR mutations, but it has a limited effect for wtEGFR NSCLC.PurposeThe current study investigated whether shikonin could enhance the antitumor effect of AZD9291 in wtEGFR NSCLC cells.MethodsSRB and colony formation assay were used to detect the proliferation of NSCLC cells, propidium iodide staining was performed to detect the apoptosis, ROS was analyzed using DCFH-DA staining, and western blot was used to detect the expression of indicated proteins.ResultsWe demonstrated that shikonin, a natural ROS inducer, could enhance the antitumor effect of AZD9291 in wtEGFR NSCLC cells. In addition, shikonin increased AZD9291-induced apoptosis accompanying with the generation of ROS and activation of ER stress. Furthermore, ROS inhibition by NAC or GSH reversed the apoptosis induced by shikonin plus AZD9291, and recovered the ER stress activated by combination treatment, indicating that ROS mediated ER stress played a vital role in this combination therapy. Moreover, shikonin increased the anticancer activity of AZD9291 in primary wtEGFR NSCLC cells through ROS-mediated ER stress.ConclusionOur study suggests that combining shikonin with AZD9291 is a promising therapeutic strategy for treating wtEGFR NSCLC patients.     

Gastrin-releasing peptide activates Akt through the epidermal growth factor receptor pathway and abrogates the effect of gefitinib

Gastrin-releasing peptide (GRP) is a mitogen for lung epithelial cells and initiates signaling through a G-protein-coupled receptor, gastrin-releasing peptide receptor (GRPR). Because GRPR transactivates the epidermal growth factor receptor (EGFR), we investigated induction by GRP of Akt, an EGFR-activated signaling pathway, and examined effects of GRP on viability of non-small cell lung carcinoma (NSCLC) cells exposed to the EGFR tyrosine kinase inhibitor gefitinib. GRP induced Akt activation primarily through c-Src-mediated transactivation of EGFR. Transfection of dominant-negative c-Src abolished GRP-induced EGFR and Akt activation. GRP induced release of amphiregulin, and pre-incubation with human amphiregulin neutralizing antibody eliminated GRP-induced Akt phosphorylation. Pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 completely blocked GRP-initiated Akt phosphorylation. These results suggest that GRP stimulates Akt activation primarily via c-Src activation, followed by extracellular release of the EGFR ligand amphiregulin, leading to the activation of EGFR and PI3K. Pretreatment of NSCLC cells with GRP resulted in an increase in the IC(50) of gefitinib of up to 9-fold; this protective effect was mimicked by the pretreatment of cells with amphiregulin and reversed by Akt or PI3K inhibition. GRP appears to rescue NSCLC cells exposed to gefitinib through release of amphiregulin and activation of the Akt pathway, suggesting GRPR and/or EGFR autocrine pathways in NSCLC cells may modulate therapeutic response to EGFR inhibitors.     

PI3K/Akt/mTOR信号通路与自噬及肿瘤的关系

自噬是一种以胞质内出现双层膜结构包裹长寿命蛋白和细胞器的自噬体为特征的细胞“自我消化”过程,在维持细胞内稳态、发育、肿瘤发生和感染中发挥重要作用。近来,诸多研究表明,自噬作为一把“双刃剑”,对肿瘤的发生发展既有促进作用,也有抑制作用。PI3K/Akt/mTOR通路由PI3激酶(PI3K)、蛋白激酶B（PKB/Akt）和哺乳动物类雷帕霉素靶蛋白（mTOR）3个作用分子组成,是一个中心的调节机构,对肿瘤细胞的生长与增殖有促进作用,同时对自噬进行抑制。本文就PI3K/Akt/mTOR通路与自噬及肿瘤发生发展的关系作一综述。     

The EphB2 tumor suppressor induces autophagic cell death via concomitant activation of the ERK1/2 and PI3K pathways

EphB2 is a tyrosine kinase receptor that has been shown to be a tumor suppressor gene in various cancers. However the mechanisms of this function are unknown. We report that EphB2 induces a form of cell death that does not involve the formation of apoptotic bodies or nuclear fragmentation and is instead accompanied by extensive vacuolization. Transmission electron microscopy demonstrates cytoplasmic vacuoles in EphB2-overexpressing cells that resembled autophagosomes. Using an EYFP-LC3 fusion protein and immunoblotting, we detected LC3 aggregation and conversion from form I to form II, both hallmarks of autophagy, in EphB2-transfected cells. Silencing of the autophagy regulating genes ATG5 or ATG7 using shRNAs, strongly prevented EphB2-induced cell death, further confirming its autophagic nature. EphB2 expression results in mitochondrial depolarization and translocation of cytochrome c from the mitochondria to the cytosol. Mapping of signaling pathways revealed novel information about the mechanisms of action of EphB2. We demonstrated that the MAPK pathway is important in the pro-death action of EphB2, through ERK1/2 phosphorylation and inhibition of this pathway using PD98059 counters EphB2-driven cell death. In addition, we found that inhibition of class III PI3K pathway, using the autophagy inhibitor 3MA, but not class I PI3K inhibition using LY294002, also effectively blocks EphB2-induced cell death. Finally, EphB2 expression inactivates Akt, which is a known inhibitor of autophagy. In conclusion, the EphB2 receptor induces an autophagic cell death that is mediated through the ERK1/2 and PI3K/Akt pathways.     

Inhibition of mTOR kinase by AZD8055 can antagonize chemotherapy-induced cell death through autophagy induction and down-regulation of p62/sequestosome 1

AZD8055 is an ATP-competitive inhibitor of mammalian target of rapamycin (mTOR) that forms two multiprotein complexes, mTORC1 and mTORC2, and negatively regulates autophagy. We demonstrate that AZD8055 stimulates and potentiates chemotherapy-mediated autophagy, as shown by LC3I-II conversion and down-regulation of the ubiquitin-binding protein p62/sequestosome 1. AZD8055-induced autophagy was pro-survival as shown by its ability to attenuate cell death and DNA damage (p-H2AX), and to enhance clonogenic survival by cytotoxic chemotherapy. Autophagy inhibition by siRNA against Beclin 1 or LC3B, or by chloroquine, partially reversed the cytoprotective effect of AZD8055 that was independent of cell cycle inhibition. The pro-survival role of autophagy was confirmed using ectopic expression of Beclin 1 that conferred cytoprotection. To determine whether autophagy-mediated down-regulation of p62/sequestosome 1 contributes to its pro-survival role, we generated p62 knockdown cells using shRNA that showed protection from chemotherapy-induced cell death and DNA damage. We also overexpressed wild-type (wt) p62 that promoted chemotherapy-induced cell death, whereas mutated p62 at functional domains (PB1, UBA) failed to do so. The ability of ectopic wt p62 to promote cell death was blocked by AZD8055. AZD8055 was shown to inhibit phosphorylation of the autophagy-initiating kinase ULK1 at Ser(757) and inhibited known targets of mTORC1 (p-mTOR Ser(2448), p70S6K, p-S6, p4EBP1) and mTORC2 (p-mTOR Ser(2481), p-AKT Ser(473)). Knockdown of mTOR, but not Raptor or Rictor, reduced p-ULK1 at Ser(757) and enhanced chemotherapy-induced autophagy that resulted in a similar cytoprotective effect as shown for AZD8055. In conclusion, AZD8055 inhibits mTOR kinase and ULK1 phosphorylation to induce autophagy whose pro-survival effect is due, in part, to down-regulation of p62.     

Targeting mTOR to Overcome Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer Cells

Aims

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however, resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells.

Methods

We selected four different types of EGFR TKI-sensitive and -resistant NSCLC cells: PC9, PC9GR, H1650 and H1975 cells as models to detect mTOR-associated signaling-pathway differences by western blot and Immunoprecipitation and evaluated the antiproliferative effect and cell cycle arrest of ku-0063794 by MTT method and flow cytometry.

Results

In the present study, we observed that mTORC2-associated Akt ser473-FOXO1 signaling pathway in a basal state was highly activated in resistant cells. In vitro mTORC1 and mTORC2 kinase activities assays showed that EGFR TKI-resistant NSCLC cell lines had higher mTORC2 kinase activity, whereas sensitive cells had higher mTORC1 kinase activity in the basal state. The ATP-competitive mTOR inhibitor ku-0063794 showed dramatic antiproliferative effects and G1-cell cycle arrest in both sensitive and resistant cells. Ku-0063794 at the IC₅₀ concentration effectively inhibited both mTOR and p70S6K phosphorylation levels; the latter is an mTORC1 substrate and did not upregulate Akt ser473 phosphorylation which would be induced by rapamycin and resulted in partial inhibition of FOXO1 phosphorylation. We also observed that EGFR TKI-sensitive and -resistant clinical NSCLC tumor specimens had higher total and phosphorylated p70S6K expression levels.

Conclusion

Our results indicate mTORC2-associated signaling-pathway was hyperactivated in EGFR TKI-resistant cells and targeting mTOR with specific mTOR inhibitors is likely a good strategy for patients with EGFR mutant NSCLC who develop EGFR TKI resistance; the potential specific roles of mTORC2 in EGFR TKI-resistant NSCLC cells were still unknown and should be further investigated.     

YM155 sensitizes non-small cell lung cancer cells to EGFR-tyrosine kinase inhibitors through the mechanism of autophagy induction

Resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as erlotinib and gefitinib, is a major clinical problem in the treatment of patients with non-small cell lung cancer (NSCLC). YM155 is a survivin small molecule inhibitor and has been demonstrated to induce cancer cell apoptosis and autophagy. EGFR-TKIs have been known to induce cancer cell autophagy. In this study, we showed that YM155 markedly enhanced the sensitivity of erlotinib to EGFR-TKI resistant NSCLC cell lines H1650 (EGFR exon 19 deletion and PTEN loss) and A549 (EGFR wild type and KRAS mutation) through inducing autophagy-dependent apoptosis and autophagic cell death. The effects of YM155 combined with erlotinib on apoptosis and autophagy inductions were more obvious than those of YM155 in combination with survivin knockdown by siRNA transfection, suggesting that YM155 induced autophagy and apoptosis in the NSCLC cells partially depend on survivin downregulation. Meanwhile, we found that the AKT/mTOR pathway is involved in modulation of survivin downregulation and autophagy induction caused by YM155. In addition, YM155 can induce DNA damage in H1650 and A549 cell lines. Moreover, combining erlotinib further augmented DNA damage by YM155, which were retarded by autophagy inhibitor 3MA, or knockdown of autophagy-related protein Beclin 1, revealing that YM155 induced DNA damage is autophagy-dependent. Similar results were also observed in vivo xenograft experiments. Therefore, combination of YM155 and erlotinib offers a promising therapeutic strategy in NSCLC with EGFR-TKI resistant phenotype.     

NT3 inhibits FGF2-induced neural progenitor cell proliferation via the PI3K/GSK3 pathway

Neurotrophin 3 (NT3), a member of the neurotrophin family, antagonizes the proliferative effect of fibroblast growth factor 2 (FGF2) on cortical precursors. However, the mechanism by which NT3 inhibits FGF2-induced neural progenitor (NP) cell proliferation is unclear. Here, using an FGF2-dependent rat neurosphere culture system, we found that NT3 inhibits both FGF2-induced neurosphere growth and bromodeoxyuridine (BrdU) incorporation in a dose-dependent manner. U0126, a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, both inhibited FGF2-induced BrdU incorporation, suggesting that the extracellular signal-regulated kinase1/2 (ERK1/2) and PI3K pathways are required for FGF2-induced NP cell proliferation. NT3 significantly inhibited FGF2-induced phosphorylation of Akt and glycogen synthase kinase 3beta (GSK3beta), a downstream kinase of Akt, whereas phosphorylation of ERK1/2 was unaffected. The inhibitory effect of NT3 on FGF2-induced NP cell proliferation was abolished by LY294002, and treatment with SB216763, a specific GSK3 inhibitor, antagonized the NT3 effect, rescuing both neurosphere growth and BrdU incorporation. Moreover, experiments with anti-NT3 antibody revealed that endogenous NT3 also plays a role in inhibiting FGF2-induced NP cell proliferation, and that anti-NT3 antibody enhanced phospho-Akt and phospho-GSK3beta levels in the presence of FGF2. These findings indicate that FGF2-induced NP cell proliferation is inhibited by NT3 via the PI3K/GSK3 pathway.     

Isoliquiritigenin Induces Apoptosis and Inhibits Xenograft Tumor Growth of Human Lung Cancer Cells by Targeting Both Wild Type and L858R/T790M Mutant EGFR

Non-small-cell lung cancer (NSCLC) is associated with diverse genetic alterations including mutation of epidermal growth factor receptor (EGFR). Isoliquiritigenin (ILQ), a chalcone derivative, possesses anticancer activities. In the present study, we investigated the effects of ILQ on the growth of tyrosine kinase inhibitor (TKI)-sensitive and -resistant NSCLC cells and elucidated its underlying mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both TKI-sensitive and -resistant NSCLC cells. ILQ-induced apoptosis was associated with the cleavage of caspase-3 and poly-(ADP-ribose)-polymerase, increased expression of Bim, and reduced expression of Bcl-2. In vitro kinase assay results revealed that ILQ inhibited the catalytic activity of both wild type and double mutant (L858R/T790M) EGFR. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wild type or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells, and attenuated the kinase activity of Akt1 and ERK2 in vitro. ILQ directly interacted with both wild type and double-mutant EGFR in an ATP-competitive manner. A docking model study showed that ILQ formed two hydrogen bonds (Glu-762 and Met-793) with wild type EGFR and three hydrogen bonds (Lys-745, Met-793, and Asp-855) with mutant EGFR. ILQ attenuated the xenograft tumor growth of H1975 cells, which was associated with decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wild type or mutant EGFR.     

Acetylshikonin induces autophagy‐dependent apoptosis through the key LKB1‐AMPK and PI3K/Akt‐regulated mTOR signalling pathways in HL‐60 cells

Acetylshikonin (ASK) is a natural naphthoquinone derivative of traditional Chinese medicine Lithospermum erythrorhyzon. It has been reported that ASK has bactericidal, anti‐inflammatory and antitumour effects. However, whether ASK induces apoptosis and autophagy in acute myeloid leukaemia (AML) cells and the underlying mechanism are still unclear. Here, we explored the roles of apoptosis and autophagy in ASK‐induced cell death and the potential molecular mechanisms in human AML HL‐60 cells. The results demonstrated that ASK remarkably inhibited the cell proliferation, viability and induced apoptosis in HL‐60 cells through the mitochondrial pathway, and ASK promoted cell cycle arrest in the S‐phase. In addition, the increased formation of autophagosomes, the turnover from light chain 3B (LC3B) I to LC3B II and decrease of P62 suggested the induction of autophagy by ASK. Furthermore, ASK significantly decreased PI3K, phospho‐Akt and p‐p70S6K expression, while enhanced phospho‐AMP‐activated protein kinase (AMPK) and phospho‐liver kinase B1(LKB1) expression. The suppression of ASK‐induced the conversion from LC3B I to LC3B II caused by the application of inhibitors of AMPK (compound C) demonstrated that ASK‐induced autophagy depends on the LKB1/AMPK pathway. These data suggested that the autophagy induced by ASK were dependent on the activation of LKB1/AMPK signalling and suppression of PI3K/Akt/mTOR pathways. The cleavage of the apoptosis‐related markers caspase‐3 and caspase‐9 and the activity of caspase‐3 induced by ASK were markedly reduced by inhibitor of AMPK (compound C), an autophagy inhibitor 3‐methyladenine (3‐MA) and another autophagy inhibitor chloroquine (CQ). Taken together, our data reveal that ASK‐induced HL‐60 cell apoptosis is dependent on the activation of autophagy via the LKB1/AMPK and PI3K/Akt‐regulated mTOR signalling pathways.     

Polyphyllin VII Induces an Autophagic Cell Death by Activation of the JNK Pathway and Inhibition of PI3K/AKT/mTOR Pathway in HepG2 Cells

Polyphyllin VII (PP7), a pennogenyl saponin isolated from Rhizoma Paridis, exhibited strong anticancer activities in various cancer types. Previous studies found that PP7 induced apoptotic cell death in human hepatoblastoma cancer (HepG2) cells. In the present study, we investigated whether PP7 could induce autophagy and its role in PP7-induced cell death, and elucidated its mechanisms. PP7 induced a robust autophagy in HepG2 cells as demonstrated by the conversion of LC3B-I to LC3B-II, degradation of P62, formation of punctate LC3-positive structures, and autophagic vacuoles tested by western blot analysis or InCell 2000 confocal microscope. Inhibition of autophagy by treating cells with autophagy inhibitor (chloroquine) abolished the cell death caused by PP7, indicating that PP7 induced an autophagic cell death in HepG2 cells. C-Jun N-terminal kinase (JNK) was activated after treatment with PP7 and pretreatment with SP600125, a JNK inhibitor, reversed PP7-induced autophagy and cell death, suggesting that JNK plays a critical role in autophagy caused by PP7. Furthermore, our study demonstrated that PP7 increased the phosphorylation of AMPK and Bcl-2, and inhibited the phosphorylation of PI3K, AKT and mTOR, suggesting their roles in the PP7-induced autophagy. This is the first report that PP7 induces an autophagic cell death in HepG2 cells via inhibition of PI3K/AKT/mTOR, and activation of JNK pathway, which induces phosphorylation of Bcl-2 and dissociation of Beclin-1 from Beclin-1/Bcl-2 complex, leading to induction of autophagy.     

Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival

The serine/threonine kinase Akt (also known as protein kinase B) is activated in response to various stimuli by a mechanism involving phosphoinositide 3-kinase (PI3-K). Akt provides a survival signal that protects cells from apoptosis induced by growth factor withdrawal, but its function in other forms of stress is less clear. Here we investigated the role of PI3-K/Akt during the cellular response to oxidant injury. H(2)O(2) treatment elevated Akt activity in multiple cell types in a time- (5-30 min) and dose (400 microM-2 mm)-dependent manner. Expression of a dominant negative mutant of p85 (regulatory component of PI3-K) and treatment with inhibitors of PI3-K (wortmannin and LY294002) prevented H(2)O(2)-induced Akt activation. Akt activation by H(2)O(2) also depended on epidermal growth factor receptor (EGFR) signaling; H(2)O(2) treatment led to EGFR phosphorylation, and inhibition of EGFR activation prevented Akt activation by H(2)O(2). As H(2)O(2) causes apoptosis of HeLa cells, we investigated whether alterations of PI3-K/Akt signaling would affect this response. Wortmannin and LY294002 treatment significantly enhanced H(2)O(2)-induced apoptosis, whereas expression of exogenous myristoylated Akt (an activated form) inhibited cell death. Constitutive expression of v-Akt likewise enhanced survival of H(2)O(2)-treated NIH3T3 cells. These results suggest that H(2)O(2) activates Akt via an EGFR/PI3-K-dependent pathway and that elevated Akt activity confers protection against oxidative stress-induced apoptosis.     

Involvement of MAP-kinase, PI3-kinase and EGF-receptor in the stimulatory effect of Neurotensin on DNA synthesis in PC3 cells

The mechanism by which neurotensin (NT) promotes the growth of prostate cancer epithelial cells is not yet defined. Here, androgen-independent PC3 cells, which express high levels of the type 1 NT-receptor (NTR1), are used to examine the involvement of epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (ERK, SAPK/JNK and p38), PI3 kinase and PKC in the mitogenic effect of NT. NT dose dependently (0.1–30 nM) enhanced phosphorylation of EGFR, ERK and Akt, reaching maximal levels within 3 min as measured by Western blotting. These effects were associated with an accumulation of EGF-like substance(s) in the medium (assayed by EGFR binding) and a 2-fold increase in DNA synthesis (assayed by [³H]thymidine incorporation). The DNA synthesis enhancement by NT was non-additive with that of EGF. The NT-induced stimulation of EGFR/ERK/Akt phosphorylation and DNA synthesis was inhibited by EGFR-tyrosine kinase inhibitors (AG1478, PD153035), metallo-endopeptidase inhibitor phosphoramidon and by heparin, but not by neutralizing anti-EGF antibody. Thus, transactivation of EGFR by NT involved heparin-binding EGF (HB-EGF or amphiregulin) rather than EGF. The effects of NT on EGFR/ERK/Akt activation and DNA synthesis were attenuated by PLC-inhibitor (U73122), PKC-inhibitors (bisindolylmaleimide, staurosporine, rottlerin), MEK inhibitor (U0126) and PI3 kinase inhibitors (wortmannin, LY 294002). We conclude that NT stimulated mitogenesis in PC3 cells by a PKC-dependent ligand-mediated transactivation of EGFR, which led to stimulation of the Raf–MEK–ERK pathway in a PI3 kinase-dependent manner.     

Mcl-1 调控NSCLC 对EGFR-TKIs敏感性的实验研究

目的:探讨人髓细胞白血病基因-1(myeloid cell leukemia-1,Mcl-1)是否参与调控非小细胞肺癌(non-small cell lung cancer,NSCLC)对EGFR-TKIs的敏感性,为非小细胞肺癌的治疗提供新的思路。方法:通过Western blot方法检测EGFR-TKIs敏感细胞H3255和耐药细胞H1975中Mcl-1蛋白的表达水平。分别给予敏感细胞H3255和耐药细胞H1975 EGFR-TKIs处理后检测细胞凋亡情况和Mcl-1蛋白表达水平。设计并合成特异性si RNA下调耐药细胞H1975中Mcl-1的表达,采用脂质体转染后通过流式细胞技术检测细胞的凋亡情况。结果:H3255细胞Mcl-1表达水平明显低于H1975细胞。一代EGFR-TKIs Gefitinib显著降低H3255细胞Mcl-1表达而不能减少H1975细胞Mcl-1表达。H1975细胞经二代EGFR-TKIs Afatinib和三代EGFR-TKIs AZD9291处理后Mcl-1表达明显减少。特异性si RNA下调H1975细胞Mcl-1表达可以促进细胞凋亡。结论:Mcl-1参与了调节NSCLC对EGFR-TKIs的敏感性,可能成为防止或逆转NSCLC对EGFR-TKIs耐药的潜在靶点。     

Inhibition of the PI3 kinase/Akt pathway enhances doxorubicin-induced apoptotic cell death in tumor cells in a p53-dependent manner

Constitutive activation of the PI3 kinase/Akt pathway is associated with the neoplastic phenotype of a large number of human tumor cells. As the anti-apoptotic role of the PI3 kinase/Akt pathway has been established, we have examined whether specific blockade of this pathway sensitizes tumor cells to DNA-damaging agent-induced cytotoxicity by enhancing apoptotic cell death. Although a PI3 kinase inhibitor, LY294002, by itself does not induce apoptotic cell death, LY294002 selectively and markedly enhances the apoptosis-inducing efficacy of doxorubicin: such an enhanced cell death is only detected in tumor cells in which the PI3 kinase/Akt pathway is constitutively activated, and it is totally dependent on the functional p53 pathway. These results suggest that the combination of a PI3 kinase/Akt pathway inhibitor and doxorubicin provides an efficient chemotherapeutic strategy for the treatment of tumor cells in which the PI3 kinase/Akt pathway is constitutively activated and the p53 pathway is functional.     

槐耳清膏抑制非小细胞肺癌细胞增殖和血管新生的作用机制研究

目的:探讨槐耳清膏抑制非小细胞肺癌(NSCLC)细胞增殖和血管新生的作用机制。方法:选择对数生长期的NSCLC细胞,经过传代培养成细胞株后采用随机法分成低剂量组、高剂量组以及空白对照组。其中低剂量组和高剂量组分别加入5μmol/L、10μmol/L的槐耳清膏进行处理,而空白对照组未加入槐耳清膏处理。利用细胞增殖毒性试验法(MTT)检测NSCLC细胞的存活率,并采用蛋白免疫印迹实验(WB)法检测表皮生长因子受体(EGFR)和血管内皮生长因子受体2(VEGFR2)、磷酸化EGFR(pEGFR)、磷酸化VEGFR2(pVEGFR2)、磷脂酰肌醇3-激酶(PI3K)、磷酸化丝氨酸/苏氨酸特异性蛋白激酶(pAKT)(pAKT)、磷酸化细胞外信号调节激酶1/2(pERK1/2)、磷酸化应激活化蛋白激酶1/2(pJNK1/2)、磷酸化-p38(pp38)、细胞周期蛋白D1(Cyclin D1)及增殖细胞核抗原(PCNA)蛋白表达水平。结果:低剂量组、高剂量组NSCLC细胞的存活率均显著低于空白对照组(P<0.05)。与空白对照组相比,槐耳清膏处理NSCLC细胞后,低剂量组、高剂量组中pEGFR、pVEGFR2蛋白的相对表达水平均显著降低(P<0.05)。与空白对照组比较,槐耳清膏处理NSCLC细胞后,低剂量组、高剂量组中PI3K蛋白、pAKT、pERK1/2、pJNK1/2、pp38、Cyclin D1及PCNA的相对表达水平均显著降低(P<0.05)。结论:基于EGFR/VEGFR2信号通路,槐耳清膏对NSCLC细胞增殖和血管新生有一定的抑制作用,可能成为一种针对NSCLC的有用靶向药物。