mTOR inhibition prevents angiotensin II-induced aortic rupture and pseudoaneurysm but promotes dissection in Apoe-deficient mice.

Aortic dissection and rupture are triggered by decreased vascular wall strength and/or increased mechanical loads. We investigated the role of mTOR signaling in aortopathy using a well-described model of angiotensin II-induced dissection, aneurysm, or rupture of the suprarenal abdominal aorta in Apoe-deficient mice. Although not widely appreciated, nonlethal hemorrhagic lesions present as pseudoaneurysms without significant dissection in this model. Angiotensin II-induced aortic tears result in free rupture, contained rupture with subadventitial hematoma (forming pseudoaneurysms), dilatation, or healing, while the media invariably thickens regardless of mural tears. Medial thickening results from smooth muscle cell hypertrophy and extracellular matrix accumulation, including matricellular proteins. Angiotensin II activates mTOR signaling in vascular wall cells, and inhibition of mTOR signaling by rapamycin prevents aortic rupture but promotes dissection. Decreased aortic rupture correlates with decreased inflammation and metalloproteinase expression, whereas extensive dissection correlates with induction of matricellular proteins that modulate adhesion of vascular cells. Thus, mTOR activation in vascular wall cells determines whether aortic tears progress to dissection or rupture. Previous mechanistic studies of aortic aneurysm and dissection by angiotensin II in Apoe-deficient mice should be reinterpreted as clinically relevant to pseudoaneurysms, and mTOR inhibition for aortic disease should be explored with caution.

Differential effect of canagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, on slow and fast skeletal muscles from nondiabetic mice.

There has been a concern that sodium-glucose cotransporter 2 (SGLT2) inhibitors could reduce skeletal muscle mass and function. Here, we examine the effect of canagliflozin (CANA), an SGLT2 inhibitor, on slow and fast muscles from nondiabetic C57BL/6J mice. In this study, mice were fed with or without CANA under ad libitum feeding, and then evaluated for metabolic valuables as well as slow and fast muscle mass and function. We also examined the effect of CANA on gene expressions and metabolites in slow and fast muscles. During SGLT2 inhibition, fast muscle function is increased, as accompanied by increased food intake, whereas slow muscle function is unaffected, although slow and fast muscle mass is maintained. When the amount of food in CANA-treated mice is adjusted to that in vehicle-treated mice, fast muscle mass and function are reduced, but slow muscle was unaffected during SGLT2 inhibition. In metabolome analysis, glycolytic metabolites and ATP are increased in fast muscle, whereas glycolytic metabolites are reduced but ATP is maintained in slow muscle during SGLT2 inhibition. Amino acids and free fatty acids are increased in slow muscle, but unchanged in fast muscle during SGLT2 inhibition. The metabolic effects on slow and fast muscles are exaggerated when food intake is restricted. This study demonstrates the differential effects of an SGLT2 inhibitor on slow and fast muscles independent of impaired glucose metabolism, thereby providing new insights into how they should be used in patients with diabetes, who are at a high risk of sarcopenia.

The therapeutic effect of hesperetin on doxorubicin-induced testicular toxicity: Potential roles of the mechanistic target of rapamycin kinase (mTOR) and dynamin-related protein 1 (DRP1).

Clinical utilization of doxorubicin (DOX), which is a commonly used chemotherapeutic, is restricted due to toxic effects on various tissues. Using hesperetin (HST), an antioxidant used in Chinese traditional medicine protects testis against DOX-induced toxicity although the molecular mechanisms are not well-known. The study was aimed to examine the possible role of the mechanistic target of rapamycin kinase (mTOR) and dynamin 1-like dynamin-related protein 1 (DRP1) in the therapeutic effects of HST on the DOX-induced testicular toxicity. Rats were divided into Control, DOX, DOX + HST, and HST groups (n = 7). Single-dose DOX (15 mg/kg) was administered intraperitoneally and HST (50 mg/kg) was administered by oral gavage every other day for 28 days. Total antioxidant status (TAS), histopathological evaluations, immunohistochemistry, and gene expression level detection analyses were performed. Histopathologically, DOX-induced testicular damage was ameliorated by HST treatment. DOX reduced testicular TAS levels and increased oxidative stress markers, 8-Hydroxy-deoxyguanosine (8-OHdG), and 4-Hydroxynonenal (4-HNE). Also, upregulated mTOR and DRP1 expressions with DOX exposure were decreased after HST treatment in the testis (p < 0.05). On the other hand, DOX-administration downregulated miR-150-5p and miR-181b-2-3p miRNAs, targeting mTOR and mRNA levels of beclin 1 (BECN1) and autophagy-related 5 (ATG5), autophagic markers. Furthermore, these levels were nearly similar to control testis samples in the DOX + HST group (p < 0.05). The study demonstrated that HST may have a therapeutic effect on DOX-induced testicular toxicity by removing reactive oxygen species (ROS) and by modulating the mTOR and DRP1 expressions, which have a critical role in regulating the balance of generation/elimination of ROS.

Rehabilitative training paired with peripheral stimulation promotes motor recovery after ischemic cerebral stroke.

Spontaneous recovery of ischemic stroke is very limited and often results in the loss of motor and sensory function. Till now, rehabilitative training is the most widely accepted therapy to improve long-term outcome. However, its effectiveness is often suboptimal, largely due to a sharp decline of neuroplasticity in adults. In this study, we hypothesized that a combination of proprioceptive stimulation and rehabilitative training will promote neuroplasticity and functional recovery post injury. To test this hypothesis, we first established a photothrombotic stroke model that lesions the hindlimb sensorimotor cortex. Next, we demonstrated that injecting Cre-dependent AAV-retro viruses into the dorsal column of PV-Cre mice achieves specific and efficient targeting of proprioceptors. With chemogenetics, this method enables chronic activation of proprioceptors. We then assessed effects of combinatorial treatment on motor and sensory functional recovery. Our results showed that pairing proprioceptive stimulation with rehabilitative training significantly promoted skilled motor, but not tactile sensory functional recovery. This further led to significant improvement when compared to rehabilitation training or proprioceptor stimulation alone. Mechanistically, combinatorial treatment promoted cortical layer V neuronal mTOR activity and sprouting of corticospinal axon into the area where proprioceptive afferents terminate in the denervated side of the spinal cord. Serving as a proof of principle, our study thus provided novel insights into the application of combining proprioceptive stimulation and rehabilitative training to improve functional recovery of ischemic stroke and other traumatic brain or spinal cord injuries.

Tumor necrosis factor-α induced protein 8 (TNFAIP8/TIPE) family is differentially expressed in oral cancer and regulates tumorigenesis through Akt/mTOR/STAT3 signaling cascade.

BACKGROUND: Highest incidence of oral cancer is reported in India with reduced survival rate in the advanced stages due to lack of effective biomarkers. Therefore, it is essential to develop novel biomarkers for the better management of this disease. In the current study, TNFAIP8/TIPE protein family comprising of four proteins is explored for its role in oral cancer. METHODS: IHC analysis of oral cancer TMA and Western blot analysis of tobacco treated oral cancer cells were performed to determine the differential expression of TIPE proteins in oral cancer. Further, CRISPR/Cas9-mediated gene editing was done to generate TIPE proteins' knockouts and MTT, colony formation, wound healing, cell cycle and Western blot analysis were performed to determine the effect of gene knockouts on various cancer hallmarks and the associated molecular targets of TIPE proteins. RESULTS AND DISCUSSION: IHC results revealed that expression of TIPE, TIPE2 and TIPE3 were upregulated and TIPE1 was downregulated in oral cancer tissues compared to normal tissues. Similar results were observed upon treating oral cancer cells with tobacco carcinogens. Furthermore, knockout of TIPE or TIPE2 or TIPE3 significantly reduced the survival, proliferation, colony formation and migration of oral cancer cells whereas knockout of TIPE1 had an opposite effect. Further, TIPE, TIPE2 and TIPE3 knockout-mediated inhibition of proliferation was associated with inhibition of cell cycle progression at S or G2/M phases, and downregulation of proteins involved in cancer progression. We found that TIPE, TIPE1 and TIPE2 proteins regulate oral cancer progression through modulation of Akt/mTOR signaling cascade, whereas TIPE3 acts through an Akt-independent mTOR/STAT3 pathway. CONCLUSION: Collectively, the TIPE proteins were proved to play significant roles in the progression of oral cancer thus warranting research and clinic attention for their therapeutic and prognostic values and raising the importance of specific targeting of TIPE proteins in cancer treatment.

Disuse muscle atrophy-improving effect of ninjin'yoeito in a mouse model.

Disuse syndrome indicates psychosomatic hypofunction caused by excess rest and motionless and muscle atrophy is termed disuse muscle atrophy. Disuse muscle atrophy-induced muscle weakness and hypoactivity further induces muscle atrophy, leading to a vicious cycle, and this is considered a factor causing secondary sarcopenia and subsequently frailty. Since frailty finally leads to a bedridden state requiring nursing, in facing a super-aging society, intervention for a risk factor of frailty, disuse muscle atrophy, is important. However, the main treatment of disuse muscle atrophy is physical therapy and there are fewer effective preventive and therapeutic drugs. The objective of this study was to search for Kampo medicine with a disuse muscle atrophy-improving effect. Ninjin'yoeito is classified as a qi-blood sohozai (dual supplement) in Chinese herbal medicine, and it has an action supplementing the spleen related to muscle. In addition, improvement of muscle mass and muscle weakness by ninjin'yoeito in a clinical study has been reported. In this study, the effect of ninjin'yoeito on disuse muscle atrophy was investigated. A disuse muscle atrophy model was prepared using male ICR mice. After surgery applying a ring for tail suspension, a 1-week recovery period was set. Ninjin'yoeito was administered by mixing it in the diet for 1 week after the recovery period, followed by tail suspension for 14 days. Ninjin'yoeito administration was continued until autopsy including the hindlimb suspension period. The mice were euthanized and autopsied immediately after completion of tail suspension, and the hindlimb muscles were collected. The food and water intakes during the hindlimb unloaded period, wet weight of the collected muscle, and muscle synthesis and muscle degradation-related factors in blood and muscle were evaluated. Ingestion of ninjin'yoeito inhibited tail suspension-induced reduction of the soleus muscle wet weight. In addition, an increase in the blood level of a muscle synthesis-related factor, IGF-1, and promotion of phosphorylation of mTOR and 4E-BP1 in the soleus muscle were observed. It was suggested that ninjin'yoeito has a disuse muscle atrophy-improving action. Promotion of the muscle synthesis pathway was considered the action mechanism of this.

Overexpressing STAMP2 attenuates diabetic renal injuries via upregulating autophagy in diabetic rats.

Diabetic nephropathy (DN) is one of the most serious and major renal complications of diabetes. Previously, Six-transmembrane Protein of Prostate 2 (STAMP2) was reported to contribute to nutritional stress. The purpose of this study is to investigate whether overexpression of STAMP2 attenuates diabetic renal injuries in DN rats. We induced the DN rat model by high-fat diet and low-dose streptozotocin and evaluated the metabolite and urine albumin/creatinine. Recombinant adeno-associated virus vectors were injected for overexpression of STAMP2. Pathophysiologic and ultrastructure features of DN by histochemical stain and transmission electron microscope, autophagy-related proteins and signaling pathway by western blotting were assessed. We found the expression of STAMP2 was decreased and autophagy was blunted in DN rat kidneys. Overexpressing STAMP2 significantly ameliorated metabolic disturbance, insulin resistance, and specifically restoring diabetic renal injury. Furthermore, overexpressing STAMP2 improved the autophagy deficiency in DN rats, as revealed by changes in the expressions of Beclin1, p62, and LC3. Furthermore, STAMP2 overexpressing promoted autophagy by inhibiting the mTOR and activating the AMPK/SIRT1 signaling pathway. Our results suggested that STAMP2 overexpression attenuated renal injuries via upregulating autophagy in DN rats. STAMP2 overexpressing promoted autophagy may been involved with inhibition of the mTOR/ULK1 and activation of the AMPK/SIRT1 signaling pathway.

Inhibition of mTOR signaling and clinical activity of metformin in oral premalignant lesions.

BACKGROUNDThe aberrant activation of the PI3K/mTOR signaling circuitry is one of the most frequently dysregulated signaling events in head and neck squamous cell carcinoma (HNSCC). Here, we conducted a single-arm, open-label phase IIa clinical trial in individuals with oral premalignant lesions (OPLs) to explore the potential of metformin to target PI3K/mTOR signaling for HNSCC prevention.METHODSIndividuals with OPLs, but who were otherwise healthy and without diabetes, underwent pretreatment and posttreatment clinical exam and biopsy. Participants received metformin for 12 weeks (week 1, 500 mg; week 2, 1000 mg; weeks 3-12, 2000 mg daily). Pretreatment and posttreatment biopsies, saliva, and blood were obtained for biomarker analysis, including IHC assessment of mTOR signaling and exome sequencing.RESULTSTwenty-three participants were evaluable for response. The clinical response rate (defined as a ≥50% reduction in lesion size) was 17%. Although lower than the proposed threshold for favorable clinical response, the histological response rate (improvement in histological grade) was 60%, including 17% complete responses and 43% partial responses. Logistic regression analysis revealed that when compared with never smokers, current and former smokers had statistically significantly increased histological responses (P = 0.016). Remarkably, a significant correlation existed between decreased mTOR activity (pS6 IHC staining) in the basal epithelial layers of OPLs and the histological (P = 0.04) and clinical (P = 0.01) responses.CONCLUSIONTo our knowledge this is the first phase II trial of metformin in individuals with OPLs, providing evidence that metformin administration results in encouraging histological responses and mTOR pathway modulation, thus supporting its further investigation as a chemopreventive agent.TRIAL REGISTRATIONNCT02581137FUNDINGNIH contract HHSN261201200031I, grants R01DE026644 and R01DE026870.

ARHGAP25 Inhibits Pancreatic Adenocarcinoma Growth by Suppressing Glycolysis via AKT/mTOR Pathway.

Increasing evidence reveals that the Rho GTPase-activating protein is a crucial negative regulator of Rho family GTPase involved in tumorigenesis. The Rho GTPase-activating protein 25 (ARHGAP25) has been shown to specifically inactivate the Rho family GTPase Rac1, which plays an important role in pancreatic adenocarcinoma (PAAD) progression. Therefore, here we aimed to clarify the expression and functional role of ARHGAP25 in PAAD. The ARHGAP25 expression was lower in PAAD tissues than that in normal pancreatic tissues based on bioinformatics analysis and immunohistochemistry staining. Overexpression of ARHGAP25 inhibited cell growth of AsPC-1 human pancreatic cancer cells in vitro, while opposite results were observed in BxPC-3 human pancreatic cancer cells with ARHGAP25 knockdown. Consistently, in vivo tumorigenicity assays also confirmed that ARHGAP25 overexpression suppressed tumor growth. Mechanically, overexpression of ARHGAP25 inactivated AKT/mTOR signaling pathway by regulating Rac1/PAK1 signaling, which was in line with the results from the Gene set enrichment analysis on The Cancer Genome Atlas dataset. Furthermore, we found that ARHGAP25 reduced HIF-1α-mediated glycolysis in PAAD cells. Treatment with PF-04691502, a dual PI3K/mTOR inhibitor, hampered the increased cell growth and glycolysis due to ARHGAP25 knockdown in PAAD cells. Altogether, these results conclude that ARHGAP25 acts as a tumor suppressor by inhibiting the AKT/mTOR signaling pathway, which might provide a therapeutic target for PAAD.

AdipoR2 inhibits human glioblastoma cell growth through the AMPK/mTOR pathway.

BACKGROUND: AdipoR2, which belongs to the seven-transmembrane-domain receptor family, has been shown to play an important role in the development of human tumours, but the underlying mechanisms are poorly understood. In this study, we found that AdipoR2 expression correlates with glioma grade. In addition, we also investigated the mechanisms behind the antiproliferative effects of AdipoR2 in U251 cells (a human glioma cell line) using colony formation and WST-8 growth assays. METHODS: The U251 cell line was cultured in vitro. Western blotting was used to detect the expression of relevant proteins. Quantitative RT-PCR was used to detect AdipoR1 and AdipoR2 expression. Flow cytometry was used to detect cell cycle assay results. The gene expression profiles of glioma samples from the CGGA database were analysed by MATLAB and GSEA software. RESULTS: The AMPK/mTOR pathway plays a central role in the regulation of cell proliferation, differentiation and migration and may promote tumorigenesis. Therefore, we can control cancer progression by modulating the AMPK/mTOR pathway. However, there is no information on the relationship between AdipoR and AMPK/mTOR in central nervous system tumours such as GBM. In this study. We found 648 upregulated genes and 436 downregulated genes correlated with AdipoR2 expression in 158 glioma samples. GSEA suggested that AdipoR2 is a cell cycle-associated gene. The results of the flow cytometry analysis indicated that AdipoR2 induced G0/G1 cell cycle arrest in U251 cells. Furthermore, we identified the AMPK/mTOR signalling axis to be involved in AdipoR2-induced cell cycle arrest. CONCLUSIONS: Our results suggest that AdipoR2 may represent a novel endogenous negative regulator of GBM cell proliferation. These findings also suggest that AdipoR2 may be a promising therapeutic target in GBM patients.

Berberine regulates the Notch1/PTEN/PI3K/AKT/mTOR pathway and acts synergistically with 17-AAG and SAHA in SW480 colon cancer cells.

CONTEXT: Berberine (BBR) is used to treat diarrhoea and gastroenteritis in the clinic. It was found to have anticolon cancer effects. OBJECTIVE: To study the anticolon cancer mechanism of BBR by connectivity map (CMAP) analysis. MATERIALS AND METHODS: CMAP based mechanistic prediction was conducted by comparing gene expression profiles of 10 µM BBR treated MCF-7 cells with that of clinical drugs such as helveticoside, ianatoside C, pyrvinium, gossypol and trifluoperazine. The treatment time was 12 h and two biological replications were performed. The DMSO-treated cells were selected as a control. The interaction between 100 µM BBR and target protein was measured by cellular thermal shift assay. The protein expression of 1-9 µM BBR treated SW480 cells were measured by WB assay. Apoptosis, cell cycle arrest, mitochondrial membrane potential (MMP) of 1-9 µM BBR treated SW480 cells were measured by flow cytometry and Hoechst 33342 staining methods. RESULTS: CMAP analysis found 14 Hsp90, HDAC, PI3K or mTOR protein inhibitors have similar functions with BBR. The experiments showed that BBR inhibited SW480 cells proliferation with IC50 of 3.436 µM, induced apoptosis, autophage, MMP depolarization and arrested G1 phase of cell cycle at 1.0 µM. BBR dose-dependently up-regulated PTEN, while inhibited Notch1, PI3K, Akt and mTOR proteins at 1.0-9.0 µM (p < 0.05). BBR also acted synergistically with Hsp90 and HDAC inhibitor (0.01 µM) in SW480 cells at 0.5 and 1.0 µM. DISCUSSION AND CONCLUSIONS: The integrative gene expression-based chemical genomic method using CMAP analysis may be applicable for mechanistic studies of other multi-targets drugs.

Upregulation of MTOR, RPS6KB1, and EIF4EBP1 in the whole blood samples of Iranian patients with multiple sclerosis compared to healthy controls.

Various genetic and epigenetic mechanisms have been suggested to play roles as the underlying pathophysiology of Multiple Sclerosis (MS). Changes in different parts of the mTOR signaling pathway are among the potential suggested mechanisms based on the specific roles of this pathway in CNS. MTOR, RPS6KB1, and EIFEBP1 genes are among important genes in the mTOR pathway, responsible for the proper function of acting proteins in this signaling pathway. This study aimed to investigate the relative expression levels of these genes in the blood samples of relapsing-remitting MS (RRMS) patients compared to healthy controls. In this case-control study blood samples were collected from 30 newly diagnosed RRMS patients and 30 age and sex-matched healthy controls. mRNA level of MTOR, RPS6KB1, and EIFEBP1 genes were assessed using Real-Time PCR. The expression of MTOR, RPS6KB1, and EIF4EBP1 genes was up regulated in MS patients compared to healthy controls (p < 0.001 for all mentioned genes). Considering gender differences, expression of the mentioned genes was increased among female patients (all P < 0.001). However, no statistically significant changes were observed among male patients. Based on the receiver operating characteristic, MTOR gene had the highest diagnostic value followed by EIF4EBP1 and RPS6KB1 genes in differentiating RRMS patients from controls. In conclusion, we found the simultaneous upregulation of MTOR, RPS6KB1, and EIF4EBP1 genes among RRMS patients. MTOR showed to have the highest diagnostic value compared to other 2 genes in differentiating RRMS patients. Further studies evaluating the importance of these findings from pharmacological and prognostic perspectives are necessary.

Decreased autophagy and fuel switching occur in a senescent hepatic cell model system.

Although aging in the liver contributes to the development of chronic liver diseases such as NAFLD and insulin resistance, little is known about the molecular and metabolic details of aging in hepatic cells. To examine these issues, we used sequential oxidative stress with hydrogen peroxide to induce premature senescence in AML12 hepatic cells. The senescent cells exhibited molecular and metabolic signatures, increased SA-ßGal and γH2A.X staining, and elevated senescence and pro-inflammatory gene expression that resembled livers from aged mice. Metabolic phenotyping showed fuel switching towards glycolysis and mitochondrial glutamine oxidation as well as impaired energy production. The senescent AML12 cells also had increased mTOR signaling and decreased autophagy which likely contributed to the fuel switching from ß-oxidation that occurred in normal AML12 cells. Additionally, senescence-associated secretory phenotype (SASP) proteins from conditioned media of senescent cells sensitized normal AML12 cells to palmitate-induced toxicity, a known pathological effect of hepatic aging. In summary, we have generated senescent AML12 cells which displayed the molecular hallmarks of aging and also exhibited the aberrant metabolic phenotype, mitochondrial function, and cell signaling that occur in the aged liver.

BMAL1 knockdown triggers different colon carcinoma cell fates by altering the delicate equilibrium between AKT/mTOR and P53/P21 pathways.

Dysregulation of the circadian timing system (CTS) frequently appears during colorectal cancer (CRC) progression. In order to better understand the role of the circadian clock in CRC progression, this study evaluated in vitro how knockdown of a core circadian protein BMAL1 (BMAL1-KD) influenced the behavior of two primary human CRC cell lines (HCT116 and SW480) and a metastatic CRC cell line (SW620).Unexpectedly, BMAL1-KD induced CRC cell-type specific responses rather than the same phenomenon throughout. First, BMAL1-KD increased AKT/mTOR activation in each CRC cell line, but to different extents. Second, BMAL1-KD-induced P53 activation varied with cell context. In a wild type P53 background, HCT116 BMAL1-KD cells quickly underwent apoptosis after shBMAL1 lentivirus transduction, while surviving cells showed less P53 but increased AKT/mTOR activation, which ultimately caused higher proliferation. In the presence of a partially functional mutant P53, SW480 BMAL1-KD cells showed moderate P53 and mTOR activation simultaneously with cell senescence. With a moderate increased AKT but unchanged mutant P53 activation, SW620 BMAL1-KD cells grew faster.Thus, under different CRC cellular pathological contexts, BMAL1 knockdown induced relatively equal effects on AKT/mTOR activation but different effects on P53 activation, which finally triggered different CRC cell fates.

The Chinese herbal prescription JZ-1 induces autophagy to protect against herpes simplex Virus-2 in human vaginal epithelial cells by inhibiting the PI3K/Akt/mTOR pathway.

ETHNOPHAMACOLOGICAL RELEVANCE: The Chinese herbal prescription JieZe-1 (JZ-1) is based on the modification of Yihuang Tang, which was first described in Fu Qingzhu Nvke by the famous Qing Dynasty doctor Shan Fu as a treatment for leukorrheal diseases. As an in-hospital preparation, JZ-1 has been used in Tongji Hospital for many years to treat various infectious diseases of the lower female genital tract, including cervicitis, vaginitis, genital herpes and condyloma acuminatum. Our previous studies have shown that JZ-1 has curative effects on Candida albicans, Trichomonas vaginalis and Ureaplasma urealyticum infections. AIM OF THE STUDY: Genital herpes is among the most common sexually transmitted diseases (STDs) worldwide and is mainly caused by herpes simplex virus type-2 (HSV-2). Current therapies can relieve symptoms in patients but do not cure or prevent the spread of the virus. This study was designed to investigate the effect of JZ-1 on HSV-2 infection and its mechanism, which is based on autophagy induction, to provide new ideas and a basis for the study of antiviral drugs. MATERIALS AND METHODS: Evaluation of the antiviral activity of JZ-1 was conducted by MTT assay and western blotting. Then, Western blot and immunofluorescence analyses, observations through transmission electron microscopy and experiments with the recombinant lentivirus vector mRFP-GFP-LC3B were used to monitor autophagic flux in VK2/E6E7 cells. To explore the mechanism by which JZ-1 regulates autophagy, western blotting and real-time quantitative PCR (qRT-PCR) were used to determine the expression of phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway proteins and to detect changes in critical molecules in the pathway after the application of a PI3K inhibitor. Additionally, the mRNA expression levels of inflammatory cytokines, namely, IL-6, IFN-α, IFN-ß and TNF-α, were measured with qRT-PCR. RESULTS: HSV-2 infection inhibited autophagy in the VK2/E6E7 cells. Further study revealed that the activation of the PI3K/Akt/mTOR pathway induced by HSV-2 infection may result in the blocked autophagic flux and inhibited autophagosome and autolysosome formation. JZ-1 exhibited significant antiviral activity in the VK2/E6E7 cells, which showed increased cell vitality and reduced viral protein expression, namely, earliest virus-specific infected cell polypeptides 5 (ICP5) and glycoprotein D (gD). We found that JZ-1 treatment inhibited the upregulation of the PI3K/Akt/mTOR pathway proteins and promoted autophagy to combat HSV-2 infection, while PI3K inhibitor pretreatment prevented the enhanced autophagy induced by JZ-1. Moreover, JZ-1 attenuated the increase in inflammatory cytokines that had been induced HSV-2 infection. CONCLUSION: Our results showed that JZ-1 protects against HSV-2 infection, and this beneficial effect may be mediated by inducing autophagy via inhibition of the PI3K/Akt/mTOR signaling axis.

Immunohistochemical Analysis of mTOR Pathway-Related Proteins in Kaposiform Hemangioendothelioma.

BACKGROUND: Mammalian target of rapamycin (mTOR) inhibitors have been shown to have excellent effects in the management of kaposiform hemangioendothelioma (KHE); however, the mechanism of action is unclear. This study identified the expressions of mTOR pathway-related proteins in different vascular tumors to provide insight into the pathogenesis of KHE. METHODS: We retrospectively reviewed the pathologic specimens of 30 patients (KHE, 15; tufted angioma [TA], 5; infantile hemangioma [IH], 5; and lymphatic malformation [LM], 5). The immunohistochemical expression of mTOR-related proteins tuberous sclerosis complex 2 (TSC2), phosphatase and tensin homologue (PTEN), phosphorylated eukaryotic translation initiation factor 4E binding protein 1 (p-4EBP1), phosphorylated mTOR (p-mTOR), and phosphorylated ribosomal protein S6 kinase B1 (p-P70S6K) were analyzed using Image-Pro Plus software. KHE had the following pattern of expression in the spindle vascular endothelial cells: TSC2 (-); PTEN (-); p-4EBP1 (+); p-mTOR (+); and p-P70S6K (+). RESULTS: All 3 patients treated with sirolimus had good responses. The TA results were similar to KHE with no significant differences (p-4EBP1: p = 0.0687; p-mTOR: p = 0.0832). The expressions of TSC2, PTEN, p-4EBP1, p-mTOR, and p-P70S6K were negative or weakly positive in IH with a statistically significant difference compared to KHE (p-4EBP1: p < 0.001; p-mTOR: p < 0.001; p-P70S6K: p < 0.001). LM had no significant differences when compared to KHE. CONCLUSIONS: The absence of TSC2 and PTEN caused abnormal activation of the mTOR signaling pathway and may be involved in the pathogenesis of KHE. The expression of mTOR-related proteins in TA and LM was similar to KHE, unlike IH. The KHE pattern of expression [PTEN (-), TSC2 (-), p-mTOR (+), p-P70S6K (+), and p-4EBP1 (+)] suggested that sirolimus may be a good therapeutic choice.

Effects of resveratrol on the expression of molecules related to the mTOR signaling pathway in pathological scar fibroblasts.

BACKGROUND: We observed the effects of resveratrol on the expression of molecules involved in the mTOR signaling pathway in pathological scar fibroblasts, including PI3K, Akt and mTOR. METHODS: We detected the expression of PI3K, Akt and mTOR in pathological scar and normal skin fibroblasts through immunofluorescence. After being treated with different concentrations of resveratrol, the expression of PI3K, Akt and mTOR mRNA and protein were detected by RT-PCR and Western Blot respectively. RESULTS: Results showed that the expression of PI3K, Akt and mTOR were significantly enhanced in pathological scar fibroblasts and mainly expressed in the nucleus, with no expression in normal skin fibroblasts. Results from RT-PCR and Western Blot tests demonstrated that after Res intervention with different concentrations for pathological scar fibroblasts, the relative expression quantity of PI3K mRNA and protein decreased and showed a dose dependent relationship. Compared to the control group, the differences were statistically significant (P<0.05). However, decrease in the expression of PI3K mRNA and protein was not obvious and there were no significant differences in comparison to the control group (P>0.05). CONCLUSIONS: The mechanism of resveratrol in the inhibition of the proliferation of pathological scar fibroblasts may be related to its down-regulation in the expression of Akt and mTOR, which are the key molecules of mTOR signaling pathway.

Mechanism of metformin enhancing the sensitivity of human pancreatic cancer cells to gem-citabine by regulating the PI3K/Akt/mTOR signaling pathway.

OBJECTIVE: To investigate the effect of metformin (MET) on enhancing the sensitivity of human pancreatic cancer cells to gemcitabine (GEM) by regulating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. MATERIALS AND METHODS: The GEM-resistant human pancreatic cancer PANC-1/GEM cell line was established, and the proliferation ability of PANC-1 and PANC-1/GEM cell lines was detected using the Cell Counting Kit-8 (CCK-8), which was then detected by flow cytometry after they were labeled by Ki67. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and Western blotting were adopted to detect the difference in the mTOR expression between PANC-1 and PANC-1/GEM cell lines. The proliferation ability of PANC-1/GEM/MET and PANC-1/GEM cell lines was determined using CCK-8 after drug-resistant cell lines were treated with 20 mmol/L MET combined with 0.4 µmol/L GEM or 0.4 µmol/L GEM alone for 48 h. Colony formation assay was applied to detect the proliferation ability of cells. The difference in the expression of mTOR/PI3K/Akt between PANC-1/GEM/MET and PANC-1/GEM cell lines was tested via qRT-PCR and Western blotting, respectively. RESULTS: Compared with PANC-1 cells, PANC-1/GEM cells had significantly enhanced proliferation ability (p<0.01). Flow cytometry results showed that the proliferation ability of PANC-1/GEM cells was notably enhanced (p<0.01). The expression level and phosphorylation level of mTOR in drug-resistant cell lines were increased (p<0.01). After the drug-resistant cell lines were treated with 20 mmol/L MET for 48 h, the proliferation ability of PANC-1/GEM/MET cells was evidently decreased compared with that of PANC-1/GEM cells (p<0.01). The messenger ribonucleic acid (mRNA) and protein expression levels of mTOR/PI3K/Akt were markedly down-regulated (p<0.01). CONCLUSIONS: MET can regulate the PI3K/Akt/mTOR signaling pathway to enhance the sensitivity of human pancreatic cancer cells to GEM.

DJ-1/PARK7 inhibits high glucose-induced oxidative stress to prevent retinal pericyte apoptosis via the PI3K/AKT/mTOR signaling pathway.

Reactive oxygen species (ROS) act through multiple pathways to induce apoptosis of retinal capillary pericytes, which is an early marker and the primary cause of the progression of diabetic retinopathy. However, the specific molecular mechanisms behind ROS-induced retinal capillary pericyte loss in diabetic retinopathy remains elusive. In this study, we investigated the molecular regulation and effects of DJ-1/PARK7 on oxidative stress and injury of rat retinal pericytes (RRPs). To perform the research, RRPs were isolated from rat retina and cultured in medium with for 2 days: control group (5.6 mM glucose), high glucose group (30 mM glucose), hypertonic group (5.6 mM glucose + 24.4 mM mannitol). We found decreased expression of DJ-1 and increased apoptosis of RRPs in high glucose group. To further study the role of DJ-1, four groups were divided as follows: normal control group (5.6 mM glucose), high glucose (30 mM glucose), empty vector control group (pcDNA3.1,30 mM glucose), DJ-1 overexpression group (pcDNA3.1-myc-DJ-1,30 mM glucose). DJ-1, P53, p-P53, cleaved caspase-3, manganese superoxide dismutase (MnSOD), catalase (CAT) and PI3K/Akt/mTOR signaling pathway in each group was detected by Western Blot. RRPs apoptosis was detected by Terminal-deoxynucleoitidyl Transferase mediated Nick End Labeling (TUNEL) and 4'6- diamidino-2-phenylindole (DAPI). Mitochondrial function was detected by jc-1 and fluorescent probes DCFH-DA was used to determine reactive oxygen species (ROS). We found that high glucose (30 mM) lasting two days can induce significant apoptosis of RRPs, increase ROS production and expressions of p-p53 and active caspase-3, impair mitochondrial function, decrease the activities of MnSOD and CAT, and decrease expression of DJ-1, p-AKT and p-mTOR. In contrast, DJ-1/PARK7 overexpression significantly increases expression of DJ-1, p-AKT and p-mTOR, increases expression and activities of MnSOD and CAT, improves mitochondrial function, decreases expression of apoptotic gene protein p-p53 and active caspase-3, reduces ROS production and reduces the apoptotic rate of RRPs induced by high glucose. These results suggest that DJ-1 may play a role in protecting RRPs from high glucose induced-oxidative injury. DJ-1 might improve mitochondrial function, inhibit ROS production and enhance antioxidant capacity to reduce apoptosis of retinal pericytes through the PI3K/AKT/mTOR signaling pathway which may be related to early pathogenesis of diabetic retinopathy.

The mTOR inhibitor everolimus overcomes CXCR4-mediated resistance to histone deacetylase inhibitor panobinostat through inhibition of p21 and mitotic regulators.

Although having promising anti-myeloma properties, the pan-histone deacetylase inhibitor (HDACi) panobinostat lacks therapeutic activity as a single agent. The aim of the current study was to elucidate the mechanisms underlying multiple myeloma (MM) resistance to panobinostat monotherapy and to define strategies to overcome it. Sensitivity of MM cell lines and primary CD138+ cells from MM patients to panobinostat correlated with reduced expression of the chemokine receptor CXCR4, whereas overexpression of CXCR4 in MM cell lines increased their resistance to panobinostat. Decreased sensitivity to HDACi was associated with reversible G0/G1 cell growth arrest while response was characterized by apoptotic cell death. Analysis of intra-cellular signaling mediators revealed the pro-survival mTOR pathway to be regulated by CXCR4 overexpression. Combining panobinostat with mTOR inhibitor everolimus abrogated the resistance to HDACi and induced synergistic cell death. The combination of panobinostat/everolimus resulted in sustained DNA damage and irreversible suppression of proliferation accompanied by robust apoptosis. Gene expression analysis revealed distinct genetic profiles of single versus combined agent exposure. Whereas panobinostat increased the expression of the cell cycle inhibitor p21, co-treatment with everolimus abrogated the increase in p21 and synergistically downregulated the expression of DNA repair genes and mitotic checkpoint regulators. Importantly, the combination of panobinostat with everolimus effectively targeted CXCR4-expressing resistant MM cells in vivo in the BM niche. In summary, our results uncover the mechanism responsible for the strong synergistic anti-MM activity of dual HDAC and mTOR inhibition and provide the rationale for a novel potential therapeutic approach to treat MM.