Qingfei Tongluo Mixture Attenuates Bleomycin-Induced Pulmonary Inflammation and Fibrosis through mTOR-Dependent Autophagy in Rats.

As an interstitial fibrosis disease characterized by diffuse alveolitis and structural alveolar disorders, idiopathic pulmonary fibrosis (IPF) has high lethality but lacks limited therapeutic drugs. A hospital preparation used for the treatment of viral pneumonia, Qingfei Tongluo mixture (QFTL), is rumored to have protective effects against inflammatory and respiratory disease. This study aims to confirm whether it has a therapeutic effect on bleomycin-induced IPF in rats and to elucidate its mechanism of action. Male SD rats were randomly divided into the following groups: control, model, CQ + QFTL (84 mg/kg chloroquine (CQ) + 3.64 g/kg QFTL), QFTL-L, M, H (3.64, 7.28, and 14.56 g/kg, respectively) and pirfenidone (PFD 420 mg/kg). After induction modeling and drug intervention, blood samples and lung tissue were collected for further detection. Body weight and lung coefficient were examined, combined with hematoxylin and eosin (H&E) and Masson staining to observe lung tissue lesions. The enzyme-linked immunosorbent assay (ELISA) and the hydroxyproline (HYP) assay kit were used to detect changes in proinflammatory factors (transforming growth factor-ß (TGF-ß), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß)) and HYP. Immunohistochemistry and Western blotting were performed to observe changes in proteins related to pulmonary fibrosis (α-smooth muscle actin (α-SMA) and matrix metalloproteinase 12 (MMP12)) and autophagy (P62 and mechanistic target of rapamycin (mTOR)). Treatment with QFTL significantly improved the adverse effects of bleomycin on body weight, lung coefficient, and pathological changes. Then, QFTL reduced bleomycin-induced increases in proinflammatory mediators and HYP. The expression changes of pulmonary fibrosis and autophagy marker proteins are attenuated by QFTL. Furthermore, the autophagy inhibitor CQ significantly reversed the downward trend in HYP levels and α-SMA protein expression, which QFTL improved in BLM-induced pulmonary fibrosis rats. In conclusion, QFTL could effectively attenuate bleomycin-induced inflammation and pulmonary fibrosis through mTOR-dependent autophagy in rats. Therefore, QFTL has the potential to be an alternative treatment for IPF in clinical practice.

Pre-heating stress associated with acute oral leucine supplementation effects in rat gastrocnemius muscle: Implications for protein synthesis signaling pathways.

Skeletal muscle is a highly plastic tissue. The role of heat shock protein 72 (Hsp72) in heat stress-induced skeletal muscle hypertrophy has been well demonstrated; however, the precise mechanisms remain unclear. Essential amino acids, such as leucine, mainly mediate muscle protein synthesis. We investigated the effects of pre-heating and increased Hsp72 expression on the mechanistic target of rapamycin (mTOR) signaling and protein synthesis following leucine administration in rat gastrocnemius muscle. To ensure increased Hsp72 expression in both the red and white portions of the muscle, one leg of male Wistar rats (10-week-old, n = 23) was heat-stressed in 43 °C water for 30 min twice at a 48-h-interval (heat-stressed leg, HS leg). The contralateral leg served as a non-heated internal control (CT leg). After the recovery period (48 h), rats were divided into the pre-administration or oral leucine administration groups. We harvested the gastrocnemius muscle (red and white parts) prior to administration and 30 and 90 min after leucine treatment (n = 7-8 per group) and intramuscular signaling responses to leucine ingestion were determined using western blotting. Heat stress significantly upregulated the expression of Hsp72 and was not altered by leucine administration. Although the phosphorylation levels of mTOR/S6K1 and ERK were similar regardless of heating, 4E-BP1 was less phosphorylated in the HS legs than the CT legs after leucine administration in the red portion of the muscles (P < 0.05). Moreover, c-Myc expression differed significantly after leucine administration in both the red and white portions of the muscles. Our findings indicate that following oral leucine administration, pre-heating partially blunted the muscle protein synthesis signaling response in the rat gastrocnemius muscle.

Caralluma pauciflorabased Ag-NPs activate ROS - induced apoptosis through down-regulation of AKT, mTOR and pI3K signaling in human gastric cancer (AGS) cells.

The phytochemicals found inCaralluma pauciflorawere studied for their ability to reduce silver nitrate in order to synthesise silver nanoparticles (AgNPs) and characterise their size and crystal structure. Thunbergol, 1,1,6-trimethyl-3-methylene-2-(3,6,9,13-tetram, Methyl nonadecanoate, Methyl cis-13,16-Docosadienate, and (1R,4aR,5S)-5-[(E)-5-Hydroxy-3-methylpent were the major compounds identified in the methanol extract by gas chromatography-mass spectrum analysis. UV/Vis spectra, Fourier-transform infrared spectroscopy, x-ray diffraction, scanning electron microscope with Energy Dispersive Xâray Analysis (EDAX), Dynamic Light Scattering (DLS) particle size analyser and atomic force microscope (AfM) were used to characterise theCaralluma paucifloraplant extract-based AgNPs. The crystal structure and estimated size of the AgNPs ranged from 20.2 to 43 nm, according to the characterization data. The anti-cancer activity of silver nanoparticles (AgNPs) synthesised fromCaralluma paucifloraextract. The AgNPs inhibited more than 60% of the AGS cell lines and had an IC50 value of 10.9640.318 g, according to the findings. The cells were further examined using fluorescence microscopy, which revealed that the AgNPs triggered apoptosis in the cells. Furthermore, the researchers looked at the levels of reactive oxygen species (ROS) in cells treated with AgNPs and discovered that the existence of ROS was indicated by green fluorescence. Finally, apoptotic gene mRNA expression analysis revealed that three target proteins (AKT, mTOR, and pI3K) were downregulated following AgNP therapy. Overall, the findings imply that AgNPs synthesised from Caralluma pauciflora extract could be used to treat human gastric cancer.

Exploring the anticancer properties of the gum of Ferula gummosa: impact on cytotoxicity, caspase 3/7 activity and apoptosis, and gene expression in SW-480 cells.

Ferula gummosa Boiss. is a well-known Iranian endemic plant that grows in the north and northeast regions of Iran. In Iranian traditional medicine, its gum is utilized to treat inflammation, pain, and infections of the gastrointestinal system. However, no studies have been conducted to investigate the anticancer potential of its gum against colorectal cancer cells. This study aimed to identify the chemical components of the gum of F. gummosa and investigate its effects on SW-480 cells. The experiments included MTT, clonogenic, micronucleus formation, acridine orange/ethidium bromide stain, DNA degradation, caspase 3/7 activity assay, and in vitro wound-healing experiment and investigating the expression of BAX, BCL2, MTOR, and PTEN genes. Chemical analysis using GC/MS identified 102 compounds. The gum had a significant cytotoxic effect on SW-480 cells, with an IC50 value of 1.8 µg/ml for 48 hours. The gum induced apoptosis. Microscopic observations revealed a decrease in cell proliferation, as evidenced by nuclear condensation, increased micronucleus formation, and inhibition of colony formation. Additionally, the gum suppressed cell migration, induced the expression of PTEN and BAX, and down-regulated MTOR and BCL2 genes. These findings suggest that Ferula gummosa has strong cytotoxic properties and warrants further investigation.

Vitamin D plays a protective role in osteoarthritis by regulating AMPK/mTOR signalling pathway to activate chondrocyte autophagy.

OBJECTIVES: The deletion of chondrocyte autophagy seems to play a key role in the pathogenesis of osteoarthritis (OA). Patients with OA often have vitamin D (VD) deficiency, and VD supplementation can improve pain and alleviate the progression of joint structures in patients. In this study, we aimed to investigate whether VD could enhance autophagy by activating the adenosine monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signalling pathway and protect against OA. METHODS: In this study, the levels of target proteins and genes were examined by western blot and qRT-PCR. Apoptotic cells were detected using TUNEL staining. Characteristics of autophagy were observed by LysoTracker red staining, mRFP-GFP-LC3 adenovirus transfection, and transmission electron microscopy. siRNA-mediated AMPK and mTOR knockdown were used to investigate the role of the AMPK/ mTOR signalling pathway in VD-induced autophagy. Haematoxylin and eosin and safranin-O/fast green staining were used detect cartilage alterations. RESULTS: We suggested that VD significantly reduced chondrocyte death and alleviated extracellular matrix degradation. Further studies showed that VD promoted the expression of the autophagy-related protein LC3II through the AMPK/mTOR signalling pathway in chondrocytes, activated lysosome activity, promoted the formation of autophagy-associated lysosomes, which played a crucial role in the degradation of intracellular organelles and maintained homeostasis. The anti-apoptotic effect of VD on chondrocytes was associated with the activation of autophagy. The group of AMPK-normal and mTOR-knockdown in the presence of VD inhibited chondrocyte apoptosis by promoting autophagy. CONCLUSIONS: This study highlights that VD can activate chondrocyte autophagy through the AMPK/mTOR signalling pathway.

Catalpol Inhibits Autophagy to Ameliorate Doxorubicin-Induced Cardiotoxicity via the AKT-mTOR Pathway.

As a kind of anthracycline, doxorubicin (DOX) is commonly used as an antitumor drug, but its clinical application has been greatly hindered due to its severe cardiotoxicity. Hence, in this study, we investigated the role of catalpol (CTP) and its effect on DOX-induced cardiotoxicity.The cardiac function of mice was evaluated by assessing lactate dehydrogenase, creatine kinase isoenzyme, heart weight to body weight, and heart weight/tibia length levels. Histopathological changes were observed using hematoxylin and eosin staining, and the terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to examine myocardial apoptosis. Superoxide dismutase (SOD) activity, glutathione (GSH), and malondialdehyde (MDA) levels were measured to confirm the changes in oxidative stress. Western blotting showed the levels of autophagy- and pathway-related proteins. Expression of autophagy marker LC3 was examined using immunofluorescence staining.CTP alleviated DOX-induced cardiac damage in mice. We further observed upregulated SOD and GSH levels, and downregulated MDA level after the CTP treatment in DOX-treated mice, indicating the protective role of CTP against oxidative injury. DOX-induced myocardial apoptosis was also inhibited by CTP treatment in mice. In addition, CTP decreased the levels of Beclin1 and LC3II/LC3I, increased the levels of P62, and activated the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway in DOX-treated mice.CTP ameliorated DOX-induced cardiotoxicity by inhibiting oxidative stress, myocardial apoptosis, and autophagy via the AKT-mTOR pathway.

Mikania micrantha extract enhances cutaneous wound healing activity through the activation of FAK/Akt/mTOR cell signaling pathway.

Mikania micrantha (MM) has been traditionally used for various health benefits, including mental health, anti-inflammatory, wound dressing, and healing of sores. However, the molecular mechanisms and dose required for the wound healing activity of MM have yet to be reported. Therefore, a study was conducted to evaluate the wound healing potential of a cold methanolic extract of MM through in vitro and in vivo studies. Human dermal fibroblast adult (HDFa) cells were treated with 0 (control), 75 ng/ml, 125 ng/ml, 250 ng/ml, and 500 ng/ml of MMmethanolic extract (MME) for 24 h. MME at 75 ng/ml has significantly (p˂0.05) promoted HDFa cell proliferation and migration. Further, MME has also been shown to enhance the invasiveness of human umbilical vascular endothelial cells (HUVECs), indicating the neovasculature for wound healing. The tube formation assay demonstrated a significant (p<0.05) increase in the angiogenic effect of the MME starting at a concentration of 75 ng/ml as compared to the control. Treatment of excision wounds in Wistar rats with 5% and 10% MME ointment significantly enhanced wound contraction compared to control animals. Incision wounds in rats treated with 5% and 10% MME showed a significant (p<0.01) increase in tensile strength compared to control. HDFa cells, and granulation tissue collected on day 14 post-wounding, revealed the modulation of the FAK/Akt/mTOR cell signaling pathway during the enhancement of wound healing. The results of gel zymography showed increased activity of MMP-2 and MMP-9 in the HDFa cells after treatment with the extract.  It is concluded that MMEcan potentially accelerate cutaneous wound healing.

MET Inhibits the Proliferation of EC Cells by Increasing MPA Sensitivity.

Context: The high resistance rate and high recurrence rate of progesterone only as a treatment for endometrial cancer (EC) limit its clinical application. Metformin (MET) may have antitumor ability. Combining MET and medroxyprogesterone acetate (MPA) may strengthen their inhibitory effects on proliferation of EC cells, but MET's mechanisms remain unclear. Objective: The study intended to identify the specific molecular mechanism that MET combined with MPA uses against EC progression. Design: The research team performed a controlled animal study. Setting: The study took place at Xuzhou Medical University in Xuzhou, China. Animals: The animals were16 female non-obese diabetic-severe combined immunodeficient (NOD-SCID) nude mice, about 12 to 16 g in weight. Interventions: The research team divided randomly, the mice into four groups and induced EC in all groups, four in each group: (1) The control group which received received normal saline, (2) the MPA group, which received 100 mg/kg of MPA; (3) the MET group, which received metformin at the rate of 200 mg/kg, each gavage volume was 0.1ml; (4) the MET+MPA group, which received 100 mg/kg of MPA and 200 mg/kg of MET. Outcome measures: The research team: (1) used a CCK-8 kit, an EdU assay, and a flow-cytometry assay to measure cancer-cell proliferation, count, and viability; determine the cell cycle; and measure apoptosis; (2) performed a Western blot analysis to determine the expression of the PR, CD133, pAkt, totalAkt, p-mTOR, and totalTOR antibodies; and (3) determined the size and volume of tumors in vivo and used immunohistochemical staining to determine expression of the Ki67 protein. Results: The MET+MPA group had a significantly lower number of cancer cells than the MET or MDA groups (both P < .001). That group also had significantly more stagnated cancer cells in the G0/G1 phase and significantly fewer cancer cells in the S phase or G2/M phase control, MET, or MPA groups (all P < .01). The MET+MPA group's PCNA and Ki-67 protein expression was significantly lower than that of the MET and MPA group. The EDU assay yielded similar results. Additionally, the MET+MPA group had significantly higher PR expression than that of to MET or MPA group (both P < .001). The MET and MPA groups' expression of CD133, p-Akt, and p-mTOR were significantly lower than those of the control group, while the MET+MPA group's levels were significantly lower than those of the MET and MPA groups. In-vivo experiments revealed that the MET and MPA groups did show decreased tumor size and volume. The MET+MPA group had tumor weights that were significantly lower and tumor volumes were significantly smaller than those of the MET and MPA groups (all P < .001). Immunohistochemical analysis revealed that the MET+MPA group's levels of the Ki-67 antigen were significantly lower than those of the MET and MPA groups. Conclusions: MET inhibited the proliferation of EC cells by increasing MPA-sensitivity, which was dependent on the inhibition of the CD133 expression and the Akt/mTOR pathway. In addition, if MET acts as an effective progestin sensitizer, it certainly offers promising therapeutic prospects for patients with early-stage EC or overgrown endometrium who have fertility requirements.

Core Target and Key Signal Pathway of Xiaoluo in the Treatment of Thyroid Cancer.

Context: At present, hormone therapy and surgery are the main treatments for thyroid cancer, and they have a quick effect but a high recurrence rate. Also, the side effects are significant. it's extremely urgent to explore treatments that can take into account both therapeutic benefits and side effects. Objective: The study intended to explore whether Xiaoluo has an inhibitory effect on the proliferation of thyroid-cancer cells in vitro and to examine the core target and key signaling pathway of Xiaoluo in the treatment of thyroid cancer, using the thyroid-cancer cell line SW579. Design: The research team performed an in-vitro study. Setting: The study took place at the College of Pharmacy at Harbin University of Commerce in Harbin, China. Outcome Measures: The research team used a Western blot analysis to detect the expression of apoptosis proteins-B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-3-and the activity related to the signaling pathways phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT)/ mammalian target of rapamycin 1 (mTORC1). The team measured optical densities and inhibition rates for the 1, 2, 5, 10, and 15 mg/mL Xiaokuo groups and for a negative control group. The research team measured apoptosis, expression of Bcl-2, Bax, and Caspase-3, and expression of P13K, AKT, and mTor for the 10 µmol/L LY294002, 10 mg/mL Xiaoluo, 100 ng/mL IGF-1, and 100 ng/mL IGF-1+10 mg/mL Xiaoluo groups and for the blank control group. Results: The inhibition of SW579 cell proliferation increased with each increase in the Xiaoluo concentration from 1-15 mg/mL, and the inhibition rate reached 49.63% when the concentration was 15 mg/ml. The Xiaoluo group's late and total apoptosis rates were significantly higher (both P < .01) than those of the blank control group. The Xiaoluo group's expression of the Bcl-2 protein was significantly lower (P < .05), and its expressions of Bax and Caspase-3 were significantly higher (both P < .01) than those of the blank control group. The Xiaoluo group's expressions of P-PI3K, P-Akt, and P-MTOR were significantly lower than those of the blank group (all P < .01). These findings were comparable to those that occurred with use of the PI3K/AKT/mTORC1 signaling pathway inhibitor LY294002. Conclusions: Xiaoluo exerts its antithyroid-cancer effects through the induction of apoptosis in thyroid cancer cells through the inhibition of the PI3K/AKT/mTORC1 signaling pathway. Xiaoluo may serve as a potential therapeutic agent for the treatment of thyroid cancer.

The roles of HDAC with IMPDH and mTOR with JAK as future targets in the treatment of rheumatoid arthritis with combination therapy.

Various studies have shown that cytokines are important regulators in rheumatoid arthritis (RA). In synovial inflammation alteration of the enzyme HDAC, IMPDH enzyme, mTOR pathway, and JAK pathway increase cytokine level. These increased cytokine levels are responsible for the inflammation in RA. Inflammation is a physiological and normal reaction of the immune system against dangerous stimuli such as injury and infection. The cytokine-based approach improves the treatment of RA. To reach this goal, various researchers and scientists are working more aggressively by using a combination approach. The present review of combination therapy provides essential evidence about the possible synergistic effect of combinatorial agents. We have focused on the effects of HDAC inhibitor with IMPDH inhibitor and mTOR inhibitor with JAK inhibitor in combination for the treatment of RA. Combining various targeted strategies can be helpful for the treatment of RA.

Low-frequency electroacupuncture improves disordered hepatic energy metabolism in insulin-resistant Zucker diabetic fatty rats via the AMPK/mTORC1/p70S6K signaling pathway.

BACKGROUND AND AIM: Disordered hepatic energy metabolism is found in obese rats with insulin resistance (IR). There are insufficient experimental studies of electroacupuncture (EA) for IR and type 2 diabetes mellitus (T2DM). The aim of this study was to probe the effect of EA on disordered hepatic energy metabolism and the adenosine monophosphate (AMP)-activated protein kinase (AMPK)/mammalian target of rapamycin complex 1 (mTORC1)/ribosomal protein S6 kinase, 70-kDa (p70S6K) signaling pathway. METHODS: Zucker Diabetic Fatty (ZDF) rats were randomly divided into three groups: EA group receiving EA treatment; Pi group receiving pioglitazone gavage; and ZF group remaining untreated (n = 8 per group). Inbred non-insulin-resistant Zucker lean rats formed an (untreated) healthy control group (ZL, n = 8). Fasting plasma glucose (FPG), fasting insulin (FINS), C-peptide, C-reactive protein (CRP) and homeostatic model assessment of insulin resistance (HOMA-IR) indices were measured. Hematoxylin-eosin (H&E) staining was used to investigate the liver morphologically. The mitochondrial structure of hepatocytes was observed by transmission electron microscopy (TEM). Western blotting was adopted to determine protein expression of insulin receptor substrate 1 (IRS-1), mTOR, mTORC1, AMPK, tuberous sclerosis 2 (TSC2) and p70S6K, and their phosphorylation. RT-PCR was used to quantify IRS-1, mTOR, mTORC1, AMPK and p70S6K mRNA levels. RESULTS: Compared with the ZF group, FPG, FINS, C-peptide, CRP and HOMA-IR levels were significantly reduced in the EA group (p < 0.05, p < 0.01). Evaluation of histopathology showed improvement in liver appearances following EA. Phosphorylation levels of AMPK, mTOR and TSC2 decreased, and IRS-1 and p70S6K increased, in hepatocytes of the ZF group, while these negative effects appeared to be alleviated by EA. CONCLUSIONS: EA can effectively ameliorate IR and regulate energy metabolism in the ZDF rat model. AMPK/mTORC1/p70S6K and related molecules may represent a potential mechanism of action underlying these effects.

Liquidambar orientalis Mill. gum extract induces autophagy via PI3K/Akt/mTOR signaling pathway in prostate cancer cells.

Liquidambar orientalis Mill (LOM), is an endemic species having a local distribution in the southwestern coastal district of Turkey. Styrax liquidus gum (SLG), is a gum-like resinous which exudates in response to injury of the trunk of LOM. The aim of the study was to investigate the cytotoxic effects and the molecular mechanisms of the ethanolic SLG extract in human prostate cancer cells. GC-MS analysis was performed to identify the volatile compound composition. Cytotoxicity was determined by XTT analysis. Apoptosis and necrosis were evaluated via ELISA assay. Autophagic cell death was detected via monodansylcadaverine (MDC) staining and by measuring the levels of LC3I and LC3II. The protein levels of p-PI3K, p-Akt and p-mTOR were evaluated by western blot analysis. In the present study, it is shown that the SLG extract containing a considerable amount of ravidomycin derivate induced autophagic cell death in prostate cancer cells via inhibiting the PI3K/Akt/mTOR pathway.

Sanse Powder Essential Oil Nanoemulsion Negatively Regulates TRPA1 by AMPK/mTOR Signaling in Synovitis: Knee Osteoarthritis Rat Model and Fibroblast-Like Synoviocyte Isolates.

Synovitis is the primary driving factor for the occurrence and development of knee osteoarthritis (KOA) and fibroblast-like synoviocytes (FLSs) and plays a crucial role during this process. Our previous works revealed that transient receptor potential ankyrin 1 (TRPA1) ion channels mediate the amplification of KOA synovitis. In recent years, essential oils have been proved to have blocking effect on transient receptor potential channels. Meanwhile, the therapeutic effect of Sanse Powder on KOA synovitis has been confirmed in clinical trials and basic studies; although, the mechanism remains unclear. In the present study, Sanse Powder essential oil nanoemulsion (SP-NEs) was prepared, and then chemical composition, physicochemical properties, and stability were investigated. Besides, both in MIA-induced KOA rats and in LPS-stimulated FLSs, we investigated whether SP-NES could alleviate KOA synovitis by interfering with AMP-activated protein kinase- (AMPK-) mammalian target of rapamycin (mTOR), an energy sensing pathway proved to negatively regulate the TRPA1. Our research shows that the top three substances in SP-NEs were tumerone, delta-cadinene, and Ar-tumerone, which accounted for 51.62% of the total, and should be considered as the main pharmacodynamic ingredient. Less inflammatory cell infiltration and type I collagen deposition were found in the synovial tissue of KOA rats treated with SP-NEs, as well as the downregulated expressions of interleukin (IL)-1ß, IL-18, and TRPA1. Besides, SP-NEs increased the phosphorylation level of AMPK and decreased the phosphorylation level of mTOR in the KOA model, and SP-NEs also upregulated expressions of peroxisome proliferator-activated receptor-gamma (PPARγ) and PPARγ coactivator-1α and downstream signaling molecules of AMPK-mTOR in vivo and in vitro. To conclude, a kind of Chinese herbal medicine for external use which is effective in treating synovitis of KOA was extracted and prepared into essential oil nanoemulsion with stable properties in the present study. It may alleviate synovitis in experimental KOA through the negative regulation of TRPA1 by AMPK-mTOR signaling.

Bufalin increases sensitivity to AKT/mTOR-induced autophagic cell death in SK-HEP-1 human hepatocellular carcinoma cells.

Bufalin is the major component of Chan-Su (a traditional Chinese medicine, TCM) extracts from the venom of Bufo bufo gargarizan. In the present study, we investigated the pharmacological mechanisms of cell cycle arrest and autophagic cell death induced by bufalin in SK-HEP-1 human hepatocellular carcinoma cells in vitro. Bufalin inhibited cell survival by MTT assay and increased cell death by trypan blue exclusion assay in a concentration-dependent manner. In addition, bufalin induced G2/M phase arrest by reducing CDK1 activity. Bufalin triggered DNA fragmentation and apoptotic cell death in SK-HEP-1 cells by DNA gel electrophoresis, TUNEL and caspase-3 activity assay, while bufalin induced autophagic cell death by double-membrane vacuoles (transmission electron microscopy, TEM), acidic vesicular organelles (acridine orange staining) and cleavage of microtubule-associated protein 1 light chain 3 (LC3). Protein expression levels of cyclin A and B, CDK1, phospho-CDK1 (Thr161), Cdc25c, phospho-Cdc25c (Ser198), phospho-AKT (Thr308), phospho-AKT (Ser473), phospho­mTOR (Ser2481) were downregulated. In contrast, protein expression levels of the Chk1, Wee1, LC3-II, Beclin-1, Atg 5, Atg 7 and Atg 12 were upregulated in SK-HEP-1 cells after bufalin treatment. Inhibition of autophagy by 3-methyladenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA) or bafilomycin A1 (an inhibitor of the vacuolar proton pump of lysosomes and endosomes) reduced the effect of bufalin on cell viability and enhanced the effect of bufalin on apoptosis. In conclusion, bufalin triggered autophagic cell death and G2/M phase arrest through the AKT/mTOR signaling pathway in SK-HEP-1 cells. Our findings showed that bufalin may be potentially efficacious in the treatment of human hepatocellular carcinoma.