Objective:To elucidate action mechanism of δ-tocotrienol in inducing apoptosis of human hepatoma HepG2 cells. Methods:Cell proliferation and viability were assessed by MTT assay; cell cycle distribution, apoptotic rate and mitochondrial membrane potential were measured by using high content screening system; the expression of apoptosis-related protein such as caspase-3, caspase-8, caspase-9, Bcl-2, Bax, tBid and cytochrome C in the HepG2 cells were evaluated by Western blotting. Results:δ-Toco-trienol inhibited HepG2 cell proliferation and induced apoptosis in a dose-dependent manner. This growth-inhibiting effect of δ-toco-trienol correlated with loss of mitochondrial membrane potential and release of cytochrome C from mitochondria to cytoplasm, and regulation of the protein expression of Bcl-2 family members, such as up-regulation of Bax and tBid and down-regulation of Bcl-2. Subsequently tocotrienol induced the activation of caspase-3, caspase-8, and caspase-9 which finally induced apoptosis of hepatoma HepG2 cells. Conclusion:δ-Tocotrienol induced apoptosis of human hepatoma HepG2 cells via mitochondrial pathway and membrane death receptor pathway.

Aim To evaluate the mechanism of HG251-induced apoptosis in K562/DOX cells.Methods Cell viability was assessed by MTT assay;cell cycle distribution,apoptosis and mitochondrial membrane potential were measured by flow cytometry;the protein expressions of P-gp,caspase-3,caspase-8,caspase-9,p53,Bcl-xL and cytochrome c in the K562/DOX cells were evaluated by Western blot.Results HG251 was able to inhibit cells proliferation,induce apoptosis,lose mitochondrial membrane potential,activate caspase-3,caspase-8,caspase-9,up-regulate p53 protein and down-regulate Bcl-xL protein in a dose-dependent manner but it had no effect on P-gp protein expression.Conclusion HG251 could overcome apoptotic resistance via up-regulating p53 protein and down-regulating Bcl-xL protein.In addition,HG251 also induced K562/DOX cells apoptosis via mitochondrial pathway and membrane death receptor pathway.

This paper is to report the study of resveratrol-induced apoptosis and its mechanisms in MCF-7 cells. MTT assay was performed to assess the cytotoxicity of resveratrol on MCF-7 cells. Hoechst 33258 staining was used to observe cellular morphologic changes in apoptosis. Apoptosis was measured by flow cytometric analysis and the protein expression was examined by Western blotting analysis. The results indicated that resveratrol could inhibit MCF-7 cell growth in a time- and concentration-dependent manner. Remarkable morphologic changes in the cells after 60 micromol L(-1) resveratrol treatment, including cell nuclear shrinkage, DNA condensation and apoptotic bodies, were observed by Hoechst 33258 staining. Resveratrol could induce apoptosis and activate p38 and p53 in a time dependent manner in MCF-7 cells. In addition, the cell growth inhibitory ratio and the apoptotic ratio of resveratrol-treated group decreased markedly by the p38 MAPK inhibitor SB203580 or p53 inhibitor pifithrin-alpha. Further experiments confirmed that resveratrol-induced p53 activation was reduced by SB203580 whereas the activation of p38 was not affected by pifithrin-alpha. In conclusion, resveratrol induced apoptosis in MCF-7 cells could be through activating p38-p53 signal pathway.

This study is to examine the effects of NNIspm-mediated cellular senescence of HepG2 cells and elucidate its potential molecular mechanism. Cellular senescence was detected with senescence-associated beta-galactosidase staining. Cell cycle distribution, intracellular fluorescence intensity and accumulation of intracellular reactive oxygen species (ROS) were detected by high content screening (HCS). Protein expression was detected by Western blotting. Polyamines content was analyzed by high performance liquid chromatography (HPLC). The results demonstrated that NNIspm significantly induced HepG2 cells senescence. This effect was due to the decrease of intracellular polyamines, the arrest at G0/G1 phase and an increase of ROS level. The molecular senescence marker p21 increased significantly after NNIspm treatment. In contrast, the protein expressions of Cyclin E and CDK2 were obvious down-regulation. The results indicated that cellular senescence induced by NNIspm was one of its antitumor mechanisms.

OBJECTIVETo study the antiproliferative effects of beta-sitosterul and its mechanism in hepatoma HepG2 cells.

METHODCell proliferation was assessed by MTT assay. Cell cycle distribution, apoptosis and mitochondrial membrane potential were measured by high content screening (HCS). The protein expression of caspase-3, caspase-8, caspase-9, Bcl-2, Bax, tBid and cytochrome c in the HepG2 cells were evaluated by Western Blots.

RESULTbeta-Sitosterul exerted significant antiproliferative effects in HepG2 cells. Furthermore, beta-sitosterul also induced HepG2 cells apoptosis, lost mitochondrial membrane potential, activated caspase-3, caspase-8 and caspase-9, up-regulate Bax, tBid protein, down-regulation Bcl-2 protein. However, beta-sitosterul had hardly any effects on QSG7701 cells.

CONCLUSIONbeta-Sitosterul exerted antiproliferative effects and induced HepG2 cells apoptosis via mitochondrial pathway and membrane death receptor pathway.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Hep G2 Cells ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Membrane Potential, Mitochondrial ; drug effects ; Sitosterols ; pharmacology

Aim To investigate the apoptotic mechanism and polyamine transporter recognition of 3-nitro-naphthalimide norspermine conjugate (NNINspm),a novel naphthalimide-polyamine conjugate, in HepG2 cells.Methods The cytotoxicity of NNINspm was assessed by MTT assay.Cell cycle distribution and apoptosis were measured by flow cytometry.The protein expression of cytochrome C,14-3-3,Bad,Bcl-xL,mTOR,p70S6K,Cdk4,p27~(kip1),Akt,Caspase-3,Caspase-9 was evaluated by Western blot.The translocation of Akt was detected by high content screening (HCS) analysis.Results NNINspm induced HepG2 cells apoptosis via Akt dephosphorylation and then triggered a series of signal events, such as Bad dephosphorylation, dissociation of 14-3-3 and Bad, and then binding to Bcl-xL,which finally resulted in mitochondrial disruption,cytochrome c release and caspase cascade activation.Furthermore,the NNINspm-mediated cell cycle arrest was due to mTOR and p70S6K dephosphorylation,Cdk4 down-regulation and p27~(kip1) up-regulation.Conclusion NNINspm induces HepG2 cell apoptosis via PI_3K/Akt signal pathway.

In the present study, the apoptotic mechanism and polyamine transporter recognition of WJH-6, a novel polyamine conjugate, were investigated in K562 and HL-60 cells. The cytotoxicity of WJH-6 was assessed by MTT assay; cell cycle distribution and apoptosis were measured by flow cytometry; the protein expression of Caspase-3, Caspase-8, Caspase-9, Bid and mitochondrial membrane potential (MMP) were evaluated by high content screening (HCS) analysis; the protein expression of cytochrome c was measured by Western blotting. The results showed that WJH-6 could be recognized and transported by polyamine transporter (PAT). Furthermore, WJH-6 was able to inhibit K562 and HL-60 cells proliferation and induce apoptosis. This apoptotic effect was relative to MMP loss, cytochrome c release from mitochondria to cytoplasm and the activation of Caspase-8, Caspase-9, Caspase-3 and Bid. These results suggested that WJH-6-induced K562 and HL-60 cells apoptosis was related with mitochondrial damage.

To develop a new small molecular probe for discovering an antitumor lead compound from the replacement of carboxylic group of two molecular antibacterial fluoroquinolones with a heterocyclic ring, a series of the C3/C3 bis-fluoroquinolones tethered with an 1, 3, 4-oxadiazole ring were synthesized as their respective HCl salts, and their structures were characterized by elemental analysis and spectral data. The in vitro antitumor activity against L1210, CHO and HL60 cell lines was also evaluated via the respective IC50 values by methylthiazole trazolium (MTT) assay.

The objective of this study is to examine the effects of ANISpm, a novel polyamine naphthalimide conjugate, with acetylsalicylic acid against hepatocellular carcinoma in vivo and in vitro and elucidate its potential molecular mechanism. The proliferation inhibition was detected by MTT assay. Cell apoptosis, intracellular fluorescence intensity and mitochondrial membrane potential (MMP) were detected by high content screening (HCS) analysis. Polyamines content was analyzed by reverse-phase high performance liquid chromatography Protein expression levels were quantified by Western blotting assay. The combination treatment strongly inhibited cell proliferation, induced cell apoptosis in HepG2 cells and H22 hepatoma cells, which was mediated by enhanced ANISpm uptake via up-regulation of spermidine/spermine N1-acetyltransferase (SSAT) and depression of intracellular polyamine. Furthermore, this synergistic apoptosis was involved in mitochondria and death-receptor signal pathway. All these findings demonstrated that the combination treatment with acetylsalicylic acid and ANISpm resulted in synergistic antitumor effects on hepatoma cells. Thus, combination therapy with these agents may be useful as a potential template for the development of better chemotherapeutic strategy against hepatoma.

Objective:To investigate the clinical efficacy of external fixators combined with anterolateral thigh musculocutaneous flap for treatment of Gustilo type IIIB/C open tibiofibular fractures.Methods:A retrospective case series study was conducted to analyze clinical data of 15 patients with Gustilo type IIIB/C open tibiofibular fractures admitted to Ruihua Hospital of Soochow University from March 2016 to June 2019. There were 11 males and 4 females, with the age of (48.5±12.6)years (range, 22-67 years). All patients underwent emergency debridement in stage I, the major blood vessels, nerves and tendons were inspected and repaired, and the fracture ends were fixed by external fixator. There were different degrees of wounds necrosis, infection and bone defect after operation. After debridement in stage II, the soft tissue defects with the dimension of 10.0 cm×5.0 cm to 30.0 cm×8.0 cm were repaired with anterolateral thigh musculocutaneous flaps whose areas ranged from 10.5 cm×5.5 cm to 30.5 cm×8.5 cm. All donor areas of the musculocutaneous flaps were sutured directly in stage I. The healing of the donor areas and musculocutaneous flaps were observed within 2 weeks after operation. At the last follow-up, the shape and sensory recovery of the flap, healing of fractures and related complications were observed. The lower extremity functional scale (LEFS) was used to evaluate the injured limb function.Results:All patients were followed-up for 12-32 months [(22.0±5.8)months]. All donor areas were healed by first intention, leaving only linear scars. The musculocutaneous flaps survived completely in all patients. Partial necrosis of large area of musculocutaneous flap occurred in 2 patients, and healed after debridement and skin grafting. Another patient had vascular crisis after musculocutaneous flap operation and survived after the embolized vein repaired by contralateral great saphenous. At the last follow-up, the shape of flap recovered well, and the feeling partially recovered with the two-point discrimination of 18-26 mm. All fractures healed well, and there were no serious infection-related complications such as osteomyelitis. The LEFS score was 47-69 points [(59.0±9.5)points].Conclusion:Theexternal fixator combined with anterolateral thigh musculocutaneous flaps for treatment of Gustilo type IIIB/Copen tibiofibular fractures can better restore the appearance of soft tissue defect of the lower leg, and can effectively reduce the occurrence of severe infection-related complications.