Decreased Akt Expression Research Articles

Catharanthine, an anticancer vinca alkaloid: an in silico and in vitro analysis of the autophagic system as the major mechanism of cell death in liver HepG2 cells.

Catharanthine, a component of the anticancer drug vinblastine along with vindoline, disrupts the cell cycle by interfering with mitotic spindle formation. Apart from their antioxidant properties, vinca alkaloids like catharanthine inhibit phosphodiesterase activity and elevate intracellular cAMP levels. The aim of this study was to investigate how catharantine affects apoptosis and autophagy.This study conducted experiments on HepG2 liver carcinoma cells with varying doses of catharanthine to evaluate cell death rates and viability and determine the IC50concentration via MTT assays. The apoptotic and autophagic effects of catharanthine were assessed using flow cytometry with annexin V and PI staining, while the expression of autophagy-related genes was analyzed through quantitative PCR. Additionally, molecular docking and molecular dynamics simulations were employed to further investigate catharanthine's impact on autophagy mechanisms.The study showed that catharanthine reduced oxidative stress and triggered apoptosis in HepG2 cells in a dose-dependent manner. Catharanthine also upregulated the expression of autophagy-related genes like LC3, Beclin1, and ULK1. Notably, catharanthine increased sirtuin-1 levels, a known autophagy inducer, while decreasing Akt expression compared to untreated cells. Molecular docking results indicated rapamycin had a stronger binding affinity with FRB (-10.7 KJ/mol-1) than catharanthine (-7.3 KJ/mol-1). Additionally, molecular dynamics simulations revealed that catharanthine interacted effectively with the FRB domain of mTOR, displaying stability and a strong binding affinity, although not as potent as rapamycin.In summary, besides its cytotoxic and pro-apoptotic effects, catharanthine activates autophagy signaling pathways and induces autophagic necrosis by inhibiting mTOR.

Direct ionizing radiation and bystander effect in mouse mesenchymal stem cells

Purpose: This study aimed to evaluate the radiation-induced direct and bystander (BYS) responses of mesenchymal stem cells (MSCs) and to characterize these cells radiobiologically. Methods and materials: MSCs were irradiated (IR) and parameters related to DNA damage and cellular signaling were verified in a dose range from 0.5 to 15 Gy; also a transwell insert co-culture system was used to study medium-mediated BYS effects. Results: The main effects on directly IR cells were seen at doses higher than 6 Gy: induction of cell death, cell cycle arrest, upregulation of p21, and alteration of redox status. Irrespective of a specific dose, induction of micronuclei formation, H2AX phosphorylation, and decreased Akt expression also occurred. Thus, mTOR expression, cell senescence, nitric oxide generation, and calcium levels, in general were not significantly modulated by radiation. Data from the linear-quadratic model showed a high alpha/beta ratio, which is consistent with a more exponential survival curve. BYS effects from the unirradiated MSCs placed into companion wells with the directly IR cells, were not observed. Conclusions: The results can be interpreted as a positive outcome, meaning that the radiation damage is restricted to the directed IR MSCs not leading to off-target cell responses.

P2RY2 Alleviates Cerebral Ischemia–Reperfusion Injury by Inhibiting YAP Phosphorylation and Reducing Mitochondrial Fission

P2Y purinoceptor 2 (P2RY2) is involved in the regulation of cell proliferation and apoptosis. The aim of this study was to explore the effects of P2RY2 on cerebral ischemia/reperfusion (I/R) injury and its molecular mechanism. Middle cerebral artery occlusion (MCAO) model in rats and OXYGEN and oxygen-glucose deprivation/reoxygenation (OGD/R) model in PC12 cells were established. P2RY2 expressions in I/R injury model in vitro and in vivo were up-regulated. In the OGD/R group, ROS level, cyto-CytC and mitochondrial fission factors expressions and cell apoptosis were increased, while SOD activity, mito-CytC and mitochondrial fusion factors expressions were decreased. P2RY2 overexpression could reverse these results. Up-regulated P2RY2 expression decreased Yes-associated protein (YAP) phosphorylation level, promote the nuclear translocation of YAP, and inhibit cell apoptosis, which can be reversed by YAP inhibitor verteporfin. The addition of PI3K/AKT inhibitor LY294002 could reverse the decrease of YAP phosphorylation level and cell apoptosis, and the increase of nuclear translocation caused by P2RY2 overexpression. Further in vivo studies validated that interference with P2RY2 increased the cerebral infarction area, decreased AKT expression, enhanced YAP phosphorylation, and inhibited the nuclear translocation of YAP. In conclusion, P2RY2 can alleviate cerebral I/R injury by inhibiting YAP phosphorylation and reducing mitochondrial fission.

Bergenin inhibits bladder cancer progression via activating the PPARγ/PTEN/Akt signal pathway.

Bladder cancer is one of the most common malignant tumors in the urinary system with high mortality and morbidity. Evidence revealed that bergenin could affect the development of cancer. Here, we aimed to investigate the effect of bergenin on bladder cancer progression and its mechanism. The effect of bergenin on cell function was first detected, followed by assessing the changes of the epithelial-mesenchymal transition (EMT) in bergenin-treated cells. The effect of bergenin on peroxisome proliferator-activated receptor γ (PPARγ)/phosphatase and tensin homolog (PTEN)/Akt signal pathway was measured by Western blotting, followed by the rescue experiments. The results showed that bergenin treatment significantly decreased cell viability and increased G1 phase arrest, accompanied by reduced expression of Ki67, cycling D1, and cycling B1 in bladder cancer cells. Apoptosis was induced by bergenin in bladder cancer cells, as evidenced by increased Bax and cleaved caspase 3 protein levels and decreased Bcl-2 level in bergenin-treated cells. Meanwhile, the inhibition of the invasion, migration, and EMT was also observed in bergenin-treated cells. Mechanism studies showed that bergenin treatment could activate PPARγ/PTEN/Akt signal pathway, as evidence by the increased nucleus PPARγ and phosphatase and tensin homolog (PTEN) expression and decreased Akt expression. Moreover, PPARγ inhibitor administration inverted the effects of bergenin on bladder cancer cell function, including the proliferation, apoptosis, invasion, and migration in bladder cancer cells. Our findings revealed that bergenin could inhibit bladder cancer progression via activating the PPARγ/PTEN/Akt signal pathway, indicating that bergenin may be a potential therapeutic medicine for bladder cancer treatment.

MicroRNA-302a promotes neointimal formation following carotid artery injury in mice by targeting PHLPP2 thus increasing Akt signaling.

The excessive proliferation and migration of smooth muscle cells (SMCs) play an important role in restenosis following percutaneous coronary interventions. MicroRNAs are able to target various genes and involved in the regulation of diverse cellular processes including cell growth and proliferation. In this study we investigated whether and how MicroRNAs regulated vascular SMC proliferation and vascular remodeling following carotid artery injury in mice. We showed that carotid artery injury-induced neointimal formation was remarkably ameliorated in microRNA (miR)-302 heterozygous mice and SMC-specific miR-302 knockout mice. In contrast, delivery of miR-302a adenovirus to the injured carotid artery enhanced neointimal formation. Upregulation of miR-302a enhanced the proliferation and migration of mouse aorta SMC (MASMC) in vitro by promoting cell cycle transition, whereas miR-302a inhibition caused the opposite results. Moreover, miR-302a promoted Akt activation by corporately decreasing Akt expression and increasing Akt phosphorylation in MASMCs. Application of the Akt inhibitor GSK690693 (5 μmol/L) counteracted the functions of miR-302a in promoting MASMC proliferation and migration. We further revealed that miR-302a directly targeted at the 3' untranslated region of PH domain and leucine rich repeat protein phosphatase 2 (PHLPP2) and negatively regulated PHLPP2 expression. Restoration of PHLPP2 abrogated the effects of miR-302a on Akt activation and MASMC motility. Furthermore, knockdown of PHLPP2 largely abolished the inhibition of neointimal formation that was observed in miR-302 heterozygous mice. Our data demonstrate that miR-302a exacerbates SMC proliferation and restenosis through increasing Akt signaling by targeting PHLPP2.

Inhibition of vitamin D analog eldecalcitol on hepatoma in vitro and in vivo.

Hepatoma is a serious liver cancer with high morbidity and mortality. Eldecalcitol (ED-71), a vitamin D analog, is extensively used as anti-cancer agent in vitro. Hepatocellular carcinoma cell, SMMC-7721 cell lines were used in this study. Transwell assay, cell apoptosis and cell cycle detection assays were investigated after treatment with ED-71 and phosphate buffered saline (PBS) as control. Sizes of tumors were measured after ED-71 treatment in a mouse model. E-cadherin and Akt gene expressions were detected by real-time PCR (RT-PCR). The results showed that cell invasion and migration were decreased markedly after ED-71 treatment compared to control group. Cell cycle detection showed that the G2 stage was 13.18% and total S-stage was 41.16% in the ED-71 group and G2 stage: 22.88%, total S-stage: 27.34% in the control group. Cell apoptosis rate was promoted in the ED-71 group. Size of the tumors reduced more after the ED-71 treatment than the PBS treatment in mice. ED-71 markedly inhibited the expression of Akt and E-cadherin, either detected by immunohistochemistry or RT-PCR. ED-71 treatment can inhibit the hepatoma agent proliferation by increasing the E-cadherin expression and decreasing Akt expression. Therefore, these findings provide novel evidence that ED-71 can be used as an anti-hepatoma agent.

Effect of Low-Intensity Pulsed Ultrasound Stimulation, Extracorporeal Shockwaves and Radial Pressure Waves on Akt, BMP-2, ERK-2, FAK and TGF-β1 During Bone Healing in Rat Tibial Defects

An experimental study was conducted to determine whether low-intensity pulsed ultrasound stimulation (LIPUS), extracorporeal shockwave treatment (ESWT) and radial pressure wave treatment (RPWT) modulate Akt, bone morphogenetic protein-2 (BMP-2), extracellular signal-regulated kinase-2 (ERK-2), focal adhesion kinase (FAK) and transforming growth factor-β1 (TGF-β1) during bone healing in rat tibial defects. Rat tibial defects were exposed to 500 shots of ESWT delivered at 0.12 mJ/mm2, 500 impulses of RPWT operated at 2.0 bar or to daily 20-min 30 mW/cm2 LIPUS. Following 1, 3 and 6 wk, bones were harvested to determine the expression and activity of Akt, BMP-2, ERK-2, FAK and TGF-β1. Animals exposed to ultrasound were followed up to 3 wk. Protein expression and activity were unchanged following LIPUS treatment. ESWT increased Akt activity 2.11-fold (p = 0.043) and TGF-β1 expression 9.11-fold (p = 0.016) at 1 wk and increased FAK activity 2.16-fold (p = 0.047) at 3 wk. RPWT increased FAK activity 2.6-fold (p = 0.028) at 3 wk and decreased Akt expression 0.52-fold (p = 0.05) at 6 wk. In conclusion, the protocols employed for ESWT and RPWT modulated distinct signaling pathways during fracture healing, while LIPUS standard protocol did not change the usual signaling pathways of the proteins investigated. Future studies are required to monitor osteogenesis so that the biologic meaning of our results can be clarified.

Ubenimex, an APN inhibitor, could serve as an anti‑tumor drug in RT112 and 5637 cells by operating in an Akt‑associated manner.

Bladder cancer, a common urinary tract tumor, has high mortality and recurrence rates associated with metastasis. Aminopeptidase N (APN) expression and metastasis have been indicated to be associated with one another. Ubenimex may function as an APN inhibitor to inhibit the degradation of the extracellular matrix during tumorigenesis. Furthermore, APN has been widely used as an adjuvant therapy for the treatment of tumors; however, little information is available regarding the impact of ubenimex on patients. Autophagy is suggested to be important in the transformation and progression of cancer. Additionally, apoptosis, which leads to the rapid demolition of cellular organelles and structures, has also been suggested as an important factor. Thus, the present study investigated the role of ubenimex in inhibiting migration and invasion by downregulating APN expression levels to induce autophagic cell death and apoptosis in bladder cancer cells. RT112 and 5637 cell lines were treated with varying doses of ubenimex. Cell viability was measured by CCK8 colorimetry and flow cytometry. Using fluorescence microscopy, autophagic cell death was assessed using acridine orange/ethidium bromide staining. Furthermore, apoptotic cell death was assessed using flow cytometry and Trypan blue staining was used to evaluate the cell death rate. Protein expression was determined by western blot analysis. Matrigel invasion assays were exploited to assess the invasion capabilities of 5637 cells. Wound-healing migration assays and Matrigel migration assays were exploited to assess the migratory abilities of 5637 cells. Treatment with ubenimex was accompanied by decreased Akt expression, indicating that ubenimex may have similar functions to Akt inhibitors. Results also indicated that ubenimex inhibited cell migration and invasion in bladder cancer cells. Furthermore, ubenimex also induced autophagic cell death and apoptosis, which suggested that mixed programmed cell death occurred in ubenimex-treated bladder cancer cells. The results from the present study suggest that ubenimex may be a potential adjuvant therapy for the treatment of bladder cancer.

Both decreased Akt expression and mTOR phosphorylation are related to decreased neuronal differentiation in the hippocampal alveus of aged mice.

Aging is an inevitable process which results in many changes. These changes are closely related to the hippocampus which is in charge of long-term learning and episodic memory. This study was to investigate age-related changes of the cell proliferation, neuroblast differentiation and Akt/mTOR signaling in the hippocampal alveus of aged mice. In the present study, we compared the differences of neurogenesis in the hippocampal alveus between adult (postnatal month 6) and aged (postnatal month 24) mice using immunohistochemistry and western blot analysis. The cell proliferation, neuroblast differentiation, and the increased astrocyte activation in the hippocampal alveus of mice were decreased in an age-dependent manner. In addition, during normal aging, the protein level of AKT, mTOR and the phosphorylation of mTOR were all decreased. However, the protein level of AKT was increased. These results indicate the neurogenesis in the immature neurons in the hippocampal alveus of aged mice was closely related to the normal aging process. In addition, during normal aging, the increased AKT phosphorylation and decreased mTOR phosphorylation in the hippocampus may play a role in aging development. The result indicates that increased activation of astrocyte, increased phosphorylation of AKT and decreased phosphorylation of mTOR may be involved in the decreased cell proliferation and neuroblast differentiation in the alveus of hippocampus of aged mice.

Apigenin inhibits cell proliferation, migration, and invasion by targeting Akt in the A549 human lung cancer cell line.

Apigenin (APG), a widely distributed flavonoid in vegetables and fruits, with low toxicity, and a nonmutagenic characteristic, has been reported to have many targets. Evidence indicates that APG can inhibit the proliferation, migration, invasion, and metastasis of some tumor cells, but the mechanism, specifically in lung cancer, is unclear. The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway regulates a diverse set of cellular functions relevant to the growth and progression of lung cancer, including proliferation, survival, migration, and invasion. Our results showed that APG exerted anti-proliferation, anti-migration, and anti-invasion effects in A549 human lung cancer cells by targeting the PI3K/Akt signaling pathway. 3-(4, 5-dimethylthiszol-2-yl)-2, 5-diphenytetrazolium bromide assay and colony formation assay showed that APG suppressed cell proliferation in a dose-dependent and time-dependent manner. Cell motility and invasiveness were assayed using a wound healing and Transwell assay, suggesting that APG inhibited the migration and invasion of A549 cells. Western blot analyses were carried out to examine the Akt signaling pathways. The results confirmed that APG decreased Akt expression and its activation. Then, cells were transfected with Akt-active and Akt-DN plasmids separately. The migration and invasion of A549 cells were significantly changed, constitutively activating Akt or knocking down Akt, indicating that APG can suppress the migration and invasion of lung cancer cells by modulating the PI3K/Akt signaling pathway. Furthermore, the results indicated that APG not only suppressed phosphorylation of Akt, thereby preventing its activation, but also inhibited its downstream gene expression of matrix metalloproteinases-9, glycogen synthase kinase-3β, and HEF1. Together, APG is a new inhibitor of Akt in lung cancer and a potential natural compound for cancer chemoprevention.

Effect of Phosphatase and Tensin Homologue on Chromosome 10 on Angiotensin II-Mediated Proliferation, Collagen Synthesis, and Akt/P27 Signaling in Neonatal Rat Cardiac Fibroblasts.

Cardiac fibroblasts (CFs) play a key role in cardiac fibrosis by regulating the balance between extracellular matrix synthesis and breakdown. Although phosphatase and tensin homologue on chromosome 10 (PTEN) has been found to play an important role in cardiovascular disease, it is not clear whether PTEN is involved in functional regulation of CFs. In the present study, PTEN was overexpressed in neonatal rat CFs via recombinant adenovirus-mediated gene transfer. The effects of PTEN overexpression on cell-cycle progression and angiotensin II- (Ang II-) mediated regulation of collagen metabolism, synthesis of matrix metalloproteinases, and Akt/P27 signaling were investigated. Compared with uninfected cells and cells infected with green fluorescent protein-expressing adenovirus (Ad-GFP), cells infected with PTEN-expressing adenovirus (Ad-PTEN) significantly increased PTEN protein and mRNA levels in CFs (P < 0.05). The proportion of CFs in the G1/S cell-cycle phase was significantly higher for PTEN-overexpressing cells. In addition, Ad-PTEN decreased mRNA expression and the protein synthesis rate of collagen types I and III and antagonized Ang II-induced collagen synthesis. Overexpression of PTEN also decreased Ang II-induced matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) production as well as gelatinase activity. Moreover, Ad-PTEN decreased Akt expression and increased P27 expression independent of Ang II stimulation. These results suggest that PTEN could regulate its functional effects in neonatal rat CFs partially via the Akt/P27 signaling pathway.

ZnT4 (SLC30A4)-null (“lethal milk”) mice have defects in mammary gland secretion and hallmarks of precocious involution during lactation

During lactation, highly specialized secretory mammary epithelial cells (MECs) produce and secrete huge quantities of nutrients and nonnutritive factors into breast milk. The zinc (Zn) transporter ZnT4 (SLC30A4) transports Zn into the trans-Golgi apparatus for lactose synthesis, and across the apical cell membrane for efflux from MECs into milk. This is consistent with observations in "lethal milk" (lm/lm) mice, which have a truncation mutation in SLC30A4, and present with not only low milk Zn concentration, but also smaller mammary glands, decreased milk volume, and lactation failure by lactation day 2. However, the molecular underpinnings of these defects are not understood. Here, we used lactating C57BL/6J(lm/lm) (ZnT4-null) mice to explore the consequences of a ZnT4-null phenotype on mammary gland function during early lactation. Lactating C57BL/6J(lm/lm) mice had significantly fewer, smaller, and collapsed alveoli comprising swollen, lipid-filled MECs during early lactation. These defects were associated with decreased Akt expression and STAT5 activation, indicative of defects in MEC secretion. In addition, increased expression of ZnT2, TNF-α, and cleaved e-cadherin concomitant with increased activation of STAT3 implicated the loss of ZnT4 in precocious activation of involution. Collectively, our study indicates that the loss of ZnT4 has profound consequences on MEC secretion and may promote tissue remodeling in the mammary gland during early lactation.

Down-regulation of Akt by methanol extracts of Impatiens balsamina L. promotes apoptosis in human oral squamous cell carcinoma cell lines.

The apoptotic activity of methanol extracts of Impatiens balsamina L. (MEIB) and related mechanisms in human oral squamous cell carcinoma (OSCC) cells have been systematically investigated. The effects of MEIB on human OSCC cell lines were investigated using trypan blue exclusion assay, MTS assay, Western blot, 4'-6-diamidino-2-phenylindole (DAPI) staining, Live/Dead assay, Immunohistochemistry, reverse transcription-polymerase chain reaction, and promoter assay. MEIB decreased cell viability and induced apoptosis in HSC-4 cells. Higher levels of p-Akt expression were observed in OSCC than in normal oral mucosa (NOM), and it correlated with poor survival of the patients. MEIB dephosphorylated p-Akt and decreased Akt expression through proteasome-dependent degradation. LY294002 (PI3K inhibitor) decreased p-Akt and Akt, resulting in enhancing MEIB-induced apoptosis. MEIB down-regulated the expression level of survivin protein at the transcriptional level and YM155 (survivin inhibitor) decreased survivin, which facilitated MEIB-induced apoptosis. MEIB and LY294002 significantly increased Bax, thereby inducing the conformational change, mitochondrial translocation, and oligomerization. In addition, MEIB-induced growth inhibition and apoptosis in OSC-20, another human OSCC cells were mediated by regulating Akt and it downstream targets, survivin and Bax. These results suggest that MEIB may serve as a potential drug candidate for the treatment of human OSCC.

Regulation of AKT gene expression by cisplatin

The use of chemotherapy drugs for the treatment of cancer is an effective therapeutic measure. However, chemoresistance affects the effectiveness of the treatment. AKT overexpression has been observed in chemoresistance. AKT expression in colon cells induced cisplatin resistance. The present study demonstrated the role of reactive oxygen species (ROS) in the induction of AKT regulation by cisplatin through the activation of JAK2/STAT3 at the transcriptional level in colon cancer cells. HCT-116 cells treated with cisplatin exhibited increased JAK2 and STAT3 activities. Reducing the expression of JAK2 in colon cancer cells using small interfering RNA (siRNA) decreased AKT expression. The present study demonstrated that AKT activation is closely associated with chemoresistance in human tumors. The inhibition of ROS decreased the levels of AKT in colon cancer cell lines. The JAK2/STAT3 pathway was also shown to mediate AKT expression and represents a potential target for overcoming cisplatin resistance in human tumors.

The role of AMPK/mTOR/S6K1 signaling axis in mediating the physiological process of exercise-induced insulin sensitization in skeletal muscle of C57BL/6 mice

The crosstalk between mTORC1/S6K1 signaling and AMPK is emerging as a powerful and highly regulated way to gauge cellular energy and nutrient content. The aim of the current study was to determine the mechanism by which exercise training reverses lipid-induced insulin resistance and the role of AMPK/mTOR/S6K1 signaling axis in mediating this response in skeletal muscle. Our results showed that high-fat feeding resulted in decreased glucose tolerance, which was associated with decreased Akt expression and increased intramuscular triglyceride deposition in the skeletal muscle of C57BL/6 mice. Impairments in lipid metabolism were accompanied by increased total protein and phosphorylation of S6K1, SREBP-1c cleavage, and decreased AMPK phosphorylation. Exercise training reversed these impairments, resulting in improved serum lipid profiles and glucose tolerance. C2C12 myotubes were exposed to palmitate, resulting in an increased insulin-dependent Akt Ser473 phosphorylation, associated with a significant increase in the level of phosphorylation of S6K1 on T389. All these changes were reversed by activation of AMPK. Consistent with this, inhibition of AMPK by compound C induced an enhanced phosphorylation of both S6K1 and Akt, and silencing of S6K1 with siRNA showed no effect on Akt phosphorylation in both the absence and presence of palmitate cultured myotubes. In addition, compound C led to an elevated SREBP-1c cleavage but was blocked by S6K1 siRNA. In summary, exercise training inhibits SREBP-1c cleavage through AMPK/mTOR/S6K1 signaling, resulting in decreased intramyocellular lipid accumulation. Our results provide new insights into the mechanism by which AMPK/mTOR/S6K1 signaling axis mediates the physiological process of exercise-induced insulin sensitization.

SiRNA-mediated Silencing of Notch-1 Enhances Docetaxel Induced Mitotic Arrest and Apoptosis in PCa Cells

Notch is an important signaling pathway that regulates cell fate, stem cell maintenance and the initiation of differentiation in many tissues. It has been reported that activation of Notch-1 contributes to tumorigenesis. However, whether Notch signaling might have a role in chemoresistance of prostate cancer is unclear. This study aimed to investigate the effects of Notch-1 silencing on the sensitivity of prostate cancer cells to docetaxel treatment. siRNA against Notch-1 was transfected into PC-3 prostate cancer cells. Proliferation, apoptosis and cell cycle distribution were examined in the presence or absence of docetaxel by MTT and flow cytometry. Expression of p21(waf1/cip1) and Akt as well as activation of Akt in PC-3 cells were detected by Western blot and Real-time PCR. Silencing of Notch-1 promoted docetaxel induced cell growth inhibition, apoptosis and cell cycle arrest in PC-3 cells. In addition, these effects were associated with increased p21(waf1/cip1) expression and decreased Akt expression and activation in PC-3 cells. Notch-1 promotes chemoresistance of prostate cancer and could be a potential therapeutic target.

Abstract A4: Withaferin A induces apoptosis in imatinib-resistant CML cells by repressing the Akt signaling pathway

Abstract Although imatinib therapy of chronic myelogenous leukemia is effective, the resistance to imatinib challenges the treatment of this disease. Therefore, search of novel drugs to overcome imatinib resistance is a critical issue in clinic. Withaferin A (WA), an extract of Withania somniferia, exhibits anticancer activity on a number of solid tumors. In this study, we investigate the effect of WA on imatinib-sensitive and -resistant CML cells. WA at low concentrations induces autophagy in imatinib- sensitive K562 cells. Co-treatment of chloroquine suppresses autophagy and switches WA-treated K562 cells to apoptosis. This data indicates that autophagy protects K562 cells from apoptosis induced by WA. However, we find that WA triggers caspase activation and apoptosis in imatinib-resistant T315I-positive cells and this effect is associated with downregulation of Akt activity. Treatment of the AKT inhibitor LY294002 also causes apoptosis in imatinib-resistant T315I- positive cells. Ectopic expression of constitutively active Akt reverses WA-induced apoptosis and caspase activation in imatinib-resistant T315I-positive cells. Molecular study demonstrates that WA represses the Akt signaling pathway by decreasing Akt expression. We find that WA causes Akt degradation through the ubiquitin- and proteasome-dependent pathway. More importantly, WA also induces AKT downregulation and apoptosis in primary CML cells. Taken together, our results suggest that imatinib-resistant T315I-positive cells are more addicted to Akt-dependent survival pathway and are more sensitive to WA. Therefore, WA can be useful for the treatment of imatinib-resistant CML. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A4.

Abstract 1098: Effect of Hypercholesterolemia on Myocardial Necrosis and Apoptosis in Response to Ischemia Reperfusion Injury

OBJECTIVE: Myocardial ischemia-reperfusion(I/R) injury often results in necrosis and apoptosis and may lead to cardiac dysfunction or death. This study investigates impact of hypercholesterolemia in the setting of I/R injury on myocardium. METHODS: The mid-left anterior descending coronary artery in normocholesterolemic (NCn=7) and hypercholesterolemic (HCn=7) Yucatan males were occluded for 60 min, followed by reperfusion for 120 min. Hemodynamic values were recorded. Monastryl blue/TTC staining was utilized to assess the area-at-risk (AAR) and necrosis. The expression of Bcl2, apoptosis inducing factor (AIF), total &amp; cleaved caspase3, total &amp; cleaved PARP, bad, BNIP3, Akt and phospho-Akt measured. The TUNEL staining utilized to assess the magnitude of apoptosis. RESULTS: Hemodynamic values of MAP (p&lt;0.01), DLVP (p&lt;0.01), +dP/pt (p&lt;0.01), and −dP/pt (p&lt;0.01) were increased 1.2 fold in HC. In HC longitudinal and horizontal segmental shortening in the AAR decreased by 49% vs. 26% (p&lt;0.01) and 68% vs. 48% (p&lt;0.01) at the end. The AAR was similar in both groups (36% vs. 34%, p=0.61) whereas infarct size increased 45% in HC (42% vs. 61%, p=0.01). The expression of anti-apoptotic Bcl2 decreased 2.3-fold (p&lt;0.01) whereas pro-apoptotic PARP and BNIP3 increased 2.8-fold (p&lt;0.01) and 1.9 fold (p&lt;0.01) respectively in HC. Ischemia decreased Akt expression 1.6-folds (p&lt;0.01) and phospho-Akt 1.5 fold (p&lt;0.01) in HC, whereas increased phospho-Akt 1.9 fold (p&lt;0.01) in NC. In HC the TUNEL+ cells increased 3.8 fold (p=0.03) in the AAR. CONCLUSIONS: This study demonstrated that hypercholesterolemia have positive ionotropic effect on myocardial function, increase infarct size in AAR, mediate pro-apoptosis and attenuate survival pathway after I/R injury.

Arsenic trioxide decreases AKT protein in a caspase-dependent manner

Arsenic trioxide (As2O3) is used clinically to treat acute promyelocytic leukemia but is less successful in other malignancies. To identify targets for potential combination therapies, we have begun to characterize signaling pathways leading to As2O3-induced cytotoxicity. Previously, we described the requirement for a reactive oxygen species-mediated, SEK1/c-Jun NH2-terminal kinase (JNK) pathway to induce apoptosis. AKT inhibits several steps in this pathway; therefore, we postulated that As2O3 might decrease its activity. Indeed, As2O3 decreases not only AKT activity but also total AKT protein, and sensitivity to As2O3 correlates with the degree of AKT protein decrease. Decreased AKT expression further correlates with JNK activation and the release of AKT from the JNK-interacting protein 1 scaffold protein known to assemble the mitogen-activated protein kinase cascade. We found that As2O3 regulates AKT protein stability without significant effects on its transcription or translation. We show that As2O3 decreases AKT protein via caspase-mediated degradation, abrogated by caspase-6, caspase-8, caspase-9, and caspase-3 inhibitors but not proteosome inhibitors. Furthermore, As2O3 enhances the ability of a heat shock protein 90 inhibitor to decrease AKT expression and increase growth inhibition. This suggests that As2O3 may be useful in combination therapies that target AKT pathways or in tumors that have constitutively active AKT expression.

Neuroprotection and free radical scavenging effects of Osmanthus fragrans

The ethanol extract of dried flowers Osmanthus fragrans (OFE) was assessed for free radical scavenging effects measured by the bleaching of the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical, scavenging of the hydroxyl anion, investigation of the ferric reducing/antioxidant power (FRAP) and lipid-peroxidation inhibition in rat tissues. OFE contained a high amount of total flavonoid and polyphenol. OFE presented the effects in the metal reducing power, FRAP assay with IC(50) values of 0.23 microg/ml, and 7.74 microg/ml, respectively. OFE presented similar activities toward the DPPH and hydroxyl anion scavenging ability with IC(50) values of 10 microg/ml. OFE with IC(50 )values between 46 and 97 microg/ml inhibited lipid peroxidation initiated by ferrous chloride in rat brain, liver, heart and kidney mitochodrias. Moreover, the neuroprotective activity of OFE was investigated under different insults (glutamate, arachidonic acid, and 6-hydroxydopamine) in Wistar rat primary cortical neurons. OFE with EC(50 )values between 66 and 165 microg/ml attenuated the neurotoxicity on MTT and LDH assays. In addition, the AKT protein expression of excitotoxicity and oxidative stress was displayed by western blotting analysis. OFE could up-regulate the glutamate and 6-OHDA decreased AKT expression. This is the first demonstration of the neuroprotective, free radical scavenging and anti-oxidative effects of O. fragrans.