Regulatory effect of autophagy on the resistance of human liver cancer cell Huh7 to lenvatinib / 中国药房

OBJECTIVE To investigate the regulatory effect of autophagy on the resistance of human liver cancer cell Huh7 to lenvatinib. METHODS Using human liver cancer cell Huh7 as subject， the lenvatinib-resist cell model （Huh7-LR） was generated by the low-dose gradient method combined with long-term administration. The sensitivity of parental cell Huh7 and drug-resistant cell Huh7-LR to lenvatinib was detected by using CCK-8 assay and flow cytometry. Western blot assay and GFP-mCherry-LC3 plasmid transfection were performed to detect the expression levels of autophagic protein Beclin-1， autophagic adapter protein sequestosome 1 （p62）， microtubule-associated protein 1 light chain 3 （LC3） and autophagic level. Furthermore， an autophagy activation model was constructed by cell starvation， the protein expression of p62 and autophagy level were detected by using Western blot assay and GFP-mCherry-LC3 plasmid transfection， and the effect of autophagy activation on the sensitivity of Huh7-LR cells to lenvatinib was detected by flow cytometry. RESULTS Compared with parental cells， the drug resistance index of Huh7-LR cells was 6.2； protein expression of p62 was increased significantly， while apoptotic rate， protein expression of Beclin-1 and LC3Ⅱ/ LC3Ⅰ ratio were all reduced significantly （P＜0.05 or P＜0.01）； the level of autophagy was decreased to some extent. Autophagy activation could significantly increase the protein expression of p62 in Huh7-LR cells （P＜0.05） and autophagy level， and significantly increase its apoptotic rate （P＜0.05）. CONCLUSIONS Autophagy is involved in lenvatinib resistance， and activating autophagy can reverse the resistance of liver cancer cells to lenvatinib to some extent.

Curcumin Induces Cycle Arrest of Colon Cancer HCT116 Cells via JAK1/STAT1/p21 Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of curcumin on the cycle arrest of human colon cancer HCT116 cells and decipher the possible molecular mechanism. MethodThe methyl thiazolyl tetrazolium （MTT） method was employed to examine the effects of curcumin （0， 12.5， 25， 50， 75， 100 μmol·L-1） and 5-fluorouracil （5-FU， 600 μmol·L-1） on the proliferation of HCT116 cells at different time points （24， 48， 72 h）. Flow cytometry was employed to examine the cycle of HCT116 cells treated with curcumin （0， 25， 50， 75 μmol·L-1） and 5-FU. Western blot was employed to determine the expression of proteins in the Janus kinase 1 （JAK1）/signal transducer and activator of transcription 1 （STAT1） /cyclin-dependent kinase inhibitor 1A （p21） pathway in HCT116 cells. The binding of STAT1 to p21 promoter region was detected by chromatin immunoprecipitation （ChIP）. Small interfering RNA （siRNA） was employed to measure the role of STAT1 in regulating the expression of p21 and that of JAK1 in regulating the activation of STAT1 by Western blot and cellular immunofluorescence， respectively. ResultCompared with the blank group， the HCT-116 cells treated with curcumin and 5-FU showed decreased viability （P<0.05）， increased proportions of cells in the G0/G1 phase （P<0.05）， decreased proportions of cells in the S phase and G2/M phase （P<0.05）， down-regulated protein level of phosphorylated p21 （P<0.05）， and up-regulated protein level of p21 （P<0.05）. Compared with the curcumin group， the p21 siRNA+ curcumin group presented decreased proportion of cells in G0/G1 phase （P<0.05）. Compared with the blank group， curcumin elevated the level of phosphorylated STAT1 （p-STAT1） （P<0.05）. Compared with the curcumin group， the curcumin + STAT1 siRNA group showcased up-regulated protein level of p21 in HCT116 cells （P<0.05）. The mechanism study showed that curcumin treatment enhanced the enrichment of STAT1 in the p21 promoter region （P<0.05） compared with the blank group. Compared with the blank group， curcumin up-regulated the level of phosphorylated JAK1 （p-JAK1） （P <0.05）. Compared with the curcumin group， the curcumin + STAT1 siRNA group demonstrated up-regulated protein levels of p-STAT1 and p21 in HCT116 cells （P<0.05）. ConclusionCurcumin may induce the cycle arrest of human colon cancer HCT116 cells by activating the JAK1/STAT1/p21 signaling pathway.

Cancer cell membrane-coated bacterial ghosts for highly efficient paclitaxel delivery against metastatic lung cancer

Chemotherapy is one of the major approaches for the treatment of metastatic lung cancer, although it is limited by the low tumor delivery efficacy of anticancer drugs. Bacterial therapy is emerging for cancer treatment due to its high immune stimulation effect; however, excessively generated immunogenicity will cause serious inflammatory response syndrome. Here, we prepared cancer cell membrane-coated liposomal paclitaxel-loaded bacterial ghosts (LP@BG@CCM) by layer-by-layer encapsulation for the treatment of metastatic lung cancer. The preparation processes were simple, only involving film formation, electroporation, and pore extrusion. LP@BG@CCM owned much higher 4T1 cancer cell toxicity than LP@BG due to its faster fusion with cancer cells. In the 4T1 breast cancer metastatic lung cancer mouse models, the remarkably higher lung targeting of intravenously injected LP@BG@CCM was observed with the almost normalized lung appearance, the reduced lung weight, the clear lung tissue structure, and the enhanced cancer cell apoptosis compared to its precursors. Moreover, several major immune factors were improved after administration of LP@BG@CCM, including the CD4+/CD8a+ T cells in the spleen and the TNF-α, IFN-γ, and IL-4 in the lung. LP@BG@CCM exhibits the optimal synergistic chemo-immunotherapy, which is a promising medication for the treatment of metastatic lung cancer.

Salidroside Regulates the miRNA-1343-3p/MAP3K6/MMP24 Signaling Molecules to Inhibit Proliferation and Invasion of Gastric Cancer Cells / 中山大学学报(医学科学版)

ObjectiveThe aim of this study is to investigate the role of salidroside in regulating the miR-1343-3p/MAP3K6 （mitogen-activated protein kinase kinase kinase 6）/MMP24 （membrane-type matrix metalloproteinase 24） signaling pathway to inhibit gastric cancer cell proliferation and migration. MethodsHuman gastric cancer cells （MGC-803） were divided into several groups based on different salidroside concentrations： a control group （0 μmol/mL）， a low-dose group （6 μmol/mL）， a medium-dose group （12 μmol/mL）， and a high-dose group （24 μmol/mL）. The anti proliferative effects of salidroside on human gastric cancer cells were evaluated by CCK-8 assay. Clonogenic assay was used to examine the effects of salidroside drugs on the clonogenic ability of human gastric cancer cells. Transwell assay was performed to detect the effect of salidroside on the invasive ability of human gastric cancer cells. Cell scratch assay was performed to detect the effect of salidroside on the migration ability of human gastric cancer cells. The miRNA expression profile was analyzed by using RNA-seq in cancer cells for 24 h after salidroside treatment. The differentially expressed miRNAs were clustered and their target genes were predicted. Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） were used to analyze and predict the functions of these target genes， and the interaction networks were established. Immunocytofluorescence was used to detect the expression of target proteins， and the transcription of candidate genes was detected by q-PCR. ResultsCCK-8 cytotoxicity experiments showed that salidroside inhibited the proliferation of MGC-803 cells （P < 0.01）. Cell cloning experiments showed that salidroside reduced the clonal formation capacity of MGC-803 cells （P < 0.000 1）. Cell invasion experiments showed that salidroside reduced the MGC-803 cell invasion capacity （P < 0.000 1）. Cell scratch experiments showed that salidroside reduced the cell migration capacity （P < 0.000 1）. RNA-seq findings showed that the expression of 44 miRNAs changed significantly after salidroside treatment in cancer cells （P < 0.05）. Bioinformatic analysis showed that there were 1 384 target mRNAs corresponding to the differentially expressed miRNAs， and the expression of the tumor suppressor miR-1343-3p was significantly upregulated after salidroside treatment （P < 0.01），and resulted in down-regulated transcription of MAP3K6 and MMP24 genes which are related to the proliferation and migration of cancer cells （P < 0.05）. Immunofluorescence experiments demonstrated that salidroside reduced protein expression levels in MAP3K6 and MMP24 genes （P < 0.000 1）. q-PCR experiments showed that salidroside reduced the mRNA expression level of MAP3K6 and MMP24 genes （P < 0.000 1）， while miRNA expression in miR-1343-3p gene was upregulated （P < 0.000 1）. ConclusionSalidroside regulates the miRNA-1343-3p/MAP3K6/MMP24 signaling molecules to inhibit proliferation and invasion of gastric cancer cells.

Real-time SERS monitoring anticancer drug release along with SERS/MR imaging for pH-sensitive chemo-phototherapy

In situ and real-time monitoring of responsive drug release is critical for the assessment of pharmacodynamics in chemotherapy. In this study, a novel pH-responsive nanosystem is proposed for real-time monitoring of drug release and chemo-phototherapy by surface-enhanced Raman spectroscopy (SERS). The Fe3O4@Au@Ag nanoparticles (NPs) deposited graphene oxide (GO) nanocomposites with a high SERS activity and stability are synthesized and labeled with a Raman reporter 4-mercaptophenylboronic acid (4-MPBA) to form SERS probes (GO-Fe3O4@Au@Ag-MPBA). Furthermore, doxorubicin (DOX) is attached to SERS probes through a pH-responsive linker boronic ester (GO-Fe3O4@Au@Ag-MPBA-DOX), accompanying the 4-MPBA signal change in SERS. After the entry into tumor, the breakage of boronic ester in the acidic environment gives rise to the release of DOX and the recovery of 4-MPBA SERS signal. Thus, the DOX dynamic release can be monitored by the real-time changes of 4-MPBA SERS spectra. Additionally, the strong T2 magnetic resonance (MR) signal and NIR photothermal transduction efficiency of the nanocomposites make it available for MR imaging and photothermal therapy (PTT). Altogether, this GO-Fe3O4@Au@Ag-MPBA-DOX can simultaneously fulfill the synergistic combination of cancer cell targeting, pH-sensitive drug release, SERS-traceable detection and MR imaging, endowing it great potential for SERS/MR imaging-guided efficient chemo-phototherapy on cancer treatment.

Correlation of OCTN2 with the chemosensitivity of prostate cancer cells to oxaliplatin / 中国药房

OBJECTIVE To study the correlation of novel organic cation transporter 2 （OCTN2） with the chemosensitivity of prostate cancer cells to oxaliplatin. METHODS Tumor samples of patients receiving radical prostatectomy were collected， and OCTN2 protein was detected with immunohistochemistry； the primary cells of the specimen were cultivated to obtain prostate cancer cell line. Inductively coupled plasma mass spectrometry was used to detect the uptake of low concentration （0.1 μmol/L） of oxaliplatin by cancer cells. Real-time PCR and Western blot were used to detect the mRNA and protein expressions of OCTN2 in cancer cells； the prostate cancer cells with the highest and lowest expression of OCTN2 protein were selected， and IC50 of oxaliplatin to prostate cancer cells was analyzed by ATP-TCA method. The inhibitory rate of plasma peak concentration of oxaliplatin （50 μmol/L） to prostate cancer cells was detected by MTT assay. Spearman method was used to analyze the relationship of the uptake of oxaliplatin by prostate cancer cells with inhibitory rate of oxaliplatin to prostate cancer cells and 505916443@qq.com mRNA expressions of OCTN2. RESULTS OCTN2 was located on the membrane of cancer cells， and the uptake of zjdtztougao@163.com oxaliplatin by cancer cells was 0.283±0.264 （n＝12）mRNA and protein expression of OCTN2 varied significantly among different cancer cells. The sensitivity of cancer cells with high expression of OCTN2 to oxaliplatin （IC50 of 4.61 μmol/L） was higher than that of cancer cells with lower expression of OCTN2 （IC50 of 26.23 μmol/L）. The inhibitory rate of oxaliplatin to cancer cells was （25.4±10.8）% （n＝12）. There was a correlation between the uptake of oxaliplatin by prostate cancer cells and the inhibition rate of oxaliplatin to prostate cancer cells and mRNA expression of OCTN2 （P＜0.05）. CONCLUSIONS High-expressed OCTN2 may promote the uptake of oxaliplatin by prostate cancer cells， and its expression can serve as a reference for predicting the sensitivity of prostate cancer cells to oxaliplatin chemotherapy.

Effects of Substrate Stiffness on Epithelial-Mesenchymal Transition of Colon Cancer Cells under Simulated Hypoxia Environment / 医用生物力学

Objective To explore the effects from the synergy of substrate stiffness and hypoxia on epithelial mesenchymal transition (EMT) of colon cancer cells SW480 by simulating the microenvironment of human colon cancer tissues. Methods Polyvinyl alcohol gels with different stiffness ( 4. 5, 20, 40 kPa) were prepared to simulate the stiffness of each part of colon cancer tissues. The morphological change of cells on substrate with different stiffness was detected under simulated hypoxia ( CoCl2 ) environment. The expression of hypoxia inducible factor (HIF-1α), and EMT markers E-cadherin, Vimentin, Snail 1 were detected by Western blot. The mRNA expression of E-cadherin, Vimentin, Snail 1, matrix metalloproteinase-2 ( MMP-2), and MMP-9 was detected by quantitative real-time PCR ( qRT-PCR). Results Under simulated hypoxia environment, with the increase of substrate stiffness, the SW480 cells spreading area increased, and transformed from round shape into irregular polygon. The EMT of SW480 could be enhanced through up-regulating expression of Vimentin, Snail 1, MMP-2, MMP-9, and down-regulating expression of E-cadherin. Conclusions This study is important for exploring the synergistic effect of substrate stiffness and hypoxia on the EMT of colon cancer cells as well as the molecular mechanism.

Cytotoxic and Apoptotic Properties of the Flavonoid-rich Ethyl Acetate Fraction of the Crude Methanol Leaf Extract of Syzygium samarangense (Blume) Merr. (Myrtaceae) against MCF-7 Breast Cancer Cells

Introduction@#Breast cancer is the most common cancer among women in the Philippines and about 3 in every 100 Filipina will be diagnosed with breast cancer in their lifetime. There is a need to discover safe, yet inexpensive herbal extracts with potential cytotoxic properties as potential treatment modalities to treat breast cancer. @*Objectives@#This study seeks to explore the cytotoxic and apoptotic properties of the ethyl acetate fraction of the defatted crude methanol leaf extract of Syzygium samarangense in MCF-7 breast cancer cell lines. @*Methods@#Screening for flavonoids of the extracts was performed using TLC, total flavonoids, total phenols, FTIR and LC-MS spectroscopy. The hydrogen peroxide and ferric reducing anti-oxidant power were used as substrates to assess in vitro anti-oxidative properties of the extracts. The MTT dye viability assay was used to assess the cytotoxic properties of the extracts against MCF-7 cells. Apoptotic properties of the extracts in MCF-7 cells were determined by caspase-3 activation assay, DNA fragmentation patterns and fluorescence microscopy after annexin-V and propidium iodide staining. @*Results@#The abundance of flavonoids in the ethyl acetate fraction of the crude methanol leaf extract was established by TLC, FTIR, LC-MS/MS, total flavonoid and total phenol analyses. The in vitro anti-oxidative properties of this extract was comparable to ascorbic acid. The median inhibitory concentration (IC50) of this extract in MCF-7 breast cancer cell lines was 7.2 mcg/mL while doxorubicin registered an IC50 of 1.2 mcg/mL. At this concentration, the extract was not cytotoxic to normally-dividing breast epithelial cells. Cytotoxicity of the extract was mediated via apoptosis as demonstrated by DNA fragmentation, caspase-3 activation and fluorescence microscopic analyses. @*Conclusion@#The study shows that the flavonoid-rich ethyl acetate fraction of the crude methanol leaf extract of S. samarangense possesses potent apoptotic and cytotoxic properties against MCF-7 breast cancer cell lines at low concentrations.

Evaluation of arjunolic acid against Brucella melitenis and in vitro cytotoxic study of lung adenocarcinomic cell line (A549)

Brucellosis, a neglected tropical disease of zoonotic nature, is caused by the genus Brucella, specifically by Brucella abortus and B. melitensis in cattle and humans, respectively. Arjunolic acid (AA) is a triterpenoid, isolated from Terminalia arjuna (Roxb.) Wight & Arn., a medicinally important plant used to treat various diseases in the Indian system of medicine. Here, we tried to evaluate AA for its antibacterial activity on Brucella and the in vitro cytotoxicity assay on human lung adenocarcinomic alveolar basal epithelial cell line (A549). Also, we assessed the synergistic effect of arjunolic acid and Tarenna asiatica (L.) Kuntze ex K.Schum. on B. melitensis. AA displayed a considerable antibacterial activity [zone of inhibition (9 mm) with a minimum inhibitory concentration of 30 ?g/mL] against B. melitensis. The rate of cell death for the cancer cells were at 100 ?g/mL concentration of AA was 82% which indicates that AA shows significant membrane disruption to cancer cells. The estimated IC50 of AA against the A549 cell line was 139.90 ?g/mL. The highest synergistic activity was exhibited forming a zone of inhibition measuring 10mm when arjunolic acid and AqE of T. asiatica was added in the concentration of 1:1, respectively.

In vitro cytotoxicity assay of Fucoidan extracted from Turbinaria conoides against cancer cell lines MCF7, A549, and normal cell line L929

Abstract The main aim of the study is to quantify the cytotoxic property of the Fucoidan extracted from the Turbinaria conoides using the MTT assay with the standard fucose. Fucoidan was extracted using the soaked water method and it was determined using the HPLC procedure the obtained Test sample Fucoidan extracted from the Turbinaria conoides and standard fucose was subjected to the cytotoxicity assay against the MCF7 Human breast cancer cell line, A549 lung cancer cell line, and L929 normal mouse fibroblast cell line. From the results it was found that the Test sample showed good IC50 value for MCF7 cell line then A549 with an increasing concentration 24 hours incubation at 37°C The IC50 for MCF7 was 115.21 µg/ml and A549 396.46µg/ml and the Fucoidan extract was checked for its cytotoxicity against the normal mouse fibroblast cell line L929, Fucoidan was found non-lethal to the L929 mouse fibroblast normal cell line. Standard fucose also gave a significant result towards MCF7 and against the L929. This indicates that the Fucoidan extracted from Tubinaria conoides shows better anticancer potential in it. Hence its application can be further extended in the pharmacological fields.

Enhancing cancer chemo-immunotherapy by biomimetic nanogel with tumor targeting capacity and rapid drug-releasing in tumor microenvironment

In the development of chemo-immunotherapy, many efforts have been focusing on designing suitable carriers to realize the co-delivery of chemotherapeutic and immunotherapeutic with different physicochemical properties and mechanisms of action. Besides, rapid drug release at the tumor site with minimal drug degradation is also essential to facilitate the antitumor effect in a short time. Here, we reported a cancer cell membrane-coated pH-responsive nanogel (NG@M) to co-deliver chemotherapeutic paclitaxel (PTX) and immunotherapeutic agent interleukin-2 (IL-2) under mild conditions for combinational treatment of triple-negative breast cancer. In the designed nanogels, the synthetic copolymer PDEA-co-HP-β-cyclodextrin-co-Pluronic F127 and charge reversible polymer dimethylmaleic anhydride-modified polyethyleneimine endowed nanogels with excellent drug-loading capacity and rapid responsive drug-releasing behavior under acidic tumor microenvironment. Benefited from tumor homologous targeting capacity, NG@M exhibited 4.59-fold higher accumulation at the homologous tumor site than heterologous cancer cell membrane-coated NG. Rapidly released PTX and IL-2 enhanced the maturation of dendritic cells and quickly activated the antitumor immune response in situ, followed by prompted infiltration of immune effector cells. By the combined chemo-immunotherapy, enhanced antitumor effect and efficient pulmonary metastasis inhibition were achieved with a prolonged median survival rate (39 days).

A novel inhibitor of N 6-methyladenosine demethylase FTO induces mRNA methylation and shows anti-cancer activities

N 6-methyladenosine (m6A) modification is critical for mRNA splicing, nuclear export, stability and translation. Fat mass and obesity-associated protein (FTO), the first identified m6A demethylase, is critical for cancer progression. Herein, we developed small-molecule inhibitors of FTO by virtual screening, structural optimization, and bioassay. As a result, two FTO inhibitors namely 18077 and 18097 were identified, which can selectively inhibit demethylase activity of FTO. Specifically, 18097 bound to the active site of FTO and then inhibited cell cycle process and migration of cancer cells. In addition, 18097 reprogrammed the epi-transcriptome of breast cancer cells, particularly for genes related to P53 pathway. 18097 increased the abundance of m6A modification of suppressor of cytokine signaling 1 (SOCS1) mRNA, which recruited IGF2BP1 to increase mRNA stability of SOCS1 and subsequently activated the P53 signaling pathway. Further, 18097 suppressed cellular lipogenesis via downregulation of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), and C/EBPβ. Animal studies confirmed that 18097 can significantly suppress in vivo growth and lung colonization of breast cancer cells. Collectively, we identified that FTO can work as a potential drug target and the small-molecule inhibitor 18097 can serve as a potential agent against breast cancer.

Effects of irbesartan combined with 5-fluorouracil on the proliferation and ERK/PPARγ signaling pathway of Lewis lung cancer cells / 中国药房

OBJECTIVE To explore the effects of irbesartan（Irb）combined with 5-fluorouracil（5-FU）on the proliferation and extracellular signal-regulated kinase （ERK）/peroxidase proliferator-activated receptor γ（PPARγ）signaling pathway of Lewis lung cancer cells. METHODS Lewis lung cancer cells from mice were divided into normal control （NC）group，Irb low-dose （LD）group（1×10－3 mmol/L），Irb high-dose （HD）group（1×10－1 mmol/L），5-FU group （10 μmol/L），Irb LD+ 5-FU group （Irb 1×10－3 mmol/L+5-FU 10 μmol/L）and Irb HD+ 5-FU group （Irb 1×10－1 mmol/L+5-FU 10 μmol/L）. MTT method was used to measure the activity of cell proliferation in each group. Plate colony formation experiment was used to determine the number of cell colonies formed in each group ；Western blot method was used to detect the expression levels of proliferating cell nuclear antigen （PCNA），p53，ERK1/2，p-ERK1/2 and PPAR γ protein in each group. RESULTS Compared with the NC group ，the cell proliferation activity ，the number of colonies formed and the protein levels of PCNA ，p-ERK1/2，and PPARγ were significantly reduced in the other five groups ，and the protein level of p 53 was significantly increased （P＜0.05）；the protein expression of ERK1/2 had no significant difference （P＞0.05）. The changes of above indexes in Irb LD+ 5-FU group and Irb HD+ 5-FU group were more significant than Irb LD group ，Irb HD group and 5-FU group （P＜0.05）. CONCLUSIONS Irb combined with 5-FU can inhibit the proliferation of Lewis lung cancer cell ，and the effect is better than that of the two alone. The mechanism may be related to the inhibition of ERK/PPARγ signal pathway.

Study of mass spectrometry imaging metabolomics of paclitaxel based on esophageal cancer multicellular tumor spheroids cultured in vitro / 药学学报

Multicellular tumor spheroids (MCTS) can simulate the structure and metabolic characteristics of tumors in vivo, which is of great significance to study the metabolic phenotype of tumor cells and the mechanism of drug intervention. In this study, esophageal cancer MCTS were constructed, and MCTS frozen sections were prepared after treated with different formulations of paclitaxel (PTX) including common PTX injection, PTX liposome and albumin bound PTX. MCTS mass spectrometry imaging analysis method was established by using air flow assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI). The visualization of the permeation and enrichment process of PTX in MCTs after PTX treatment was realized, and the spatially resolved metabolomics of PTX injection group was studied. The results showed that the permeation and enrichment behavior of PTX in MCTs model were related to the formulations. The changes of endogenous metabolites in MCTs of esophageal cancer after treated with PTX injection had temporal and spatial characteristics. The metabolic changes of MCTS during the initial 0-4 hours were dominated by the down-regulation of middle-high polarity metabolites and some lipids in the central region of MCTS, while the metabolic changes of MCTS during 8-72 hours were mainly up-regulated by lipid metabolites in the peripheral region of MCTS. The combination of in vivo tumor-associated MCTs model with label free, highly sensitive and high coverage mass spectrometry imaging technology provided a new method and strategy for the study of pharmacometabolomics.

Isobavachalcone induces cell death through multiple pathways in human breast cancer MCF-7 cells / 南方医科大学学报

OBJECTIVE@#To explore the effects of isobavachalcone (IBC) on cell death of human breast cancer MCF-7 cells and explore the possible mechanism.@*METHODS@#MCF-7 cells were treated with different concentrations of IBC, and the changes in cell proliferation were assessed using MTT assay. Apoptosis of MCF-7 cells following treatment with 10, 20, and 40 μmol/L IBC was analyzed using flow cytometry with annexin V-FITC/PI double staining and fluorescence microscopy, and the expressions of apoptosis- and autophagy-related proteins (Bax, Bcl-2, Akt, p-Akt, p62, and LC3) were detected with Western blotting. Electron microscopy was used to observe the changes in submicrostructure of the cells following treatment with 40 μmol/L IBC. JC-1 assay kit, ATP assay kit, and reactive oxygen species (ROS) kit were used to determine the effect of IBC on mitochondrial function of the cells.@*RESULTS@#MTT assay showed that IBC significantly inhibited the proliferation of MCF-7 cells in a concentration- and time-dependent manner, with IC50 values of 38.46, 31.31, and 28.26 μmol/L at 24, 48, and 72 h, respectively. IBC also concentration-dependently induced apoptosis of MCF-7 cells. IBC-induced cell death was inhibited by z-VAD-fmk, a caspase inhibitor (P < 0.05), but not by the necroptosis inhibitor necrostatin-1 (Nec-1). Western blotting showed that IBC-induced MCF-7 cell apoptosis by increasing Bax expression and down-regulating the expressions of Bcl-2, Akt and p-Akt-473 (all P < 0.05). With the increase of IBC concentration, the expression of autophagy-related protein p62 and the LC3-II/I ratio increased progressively. Electron microscopy revealed the presence of autophagic bodies in IBC-treated MCF-7 cells. IBC treatment also resulted in decreased mitochondrial membrane potential and intracellular ATP level and increased ROS accumulation in MCF-7 cells (P < 0.05).@*CONCLUSION@#IBC is capable of inducing both apoptosis and autophagy in MCF-7 cells, suggesting the potential value of IBC as a lead compound in the development of anti-breast cancer agents.

Betulinic Acid Induces Autophagy and Apoptosis of Human Colorectal Cancer SW620 Cells by Regulating PI3K/Akt/mTOR Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of betulinic acid （BA） on apoptosis and autophagy of human colorectal cancer SW620 cells and the regulatory role of phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway. MethodCell viability was detected by methyl thiazolyl tetrazolium （MTT） colorimetry to determine the optimal administration time and dosage for subsequent experiments. Four groups were designed， including blank group and low-， medium-， and high-dose BA groups. Hematoxylin-eosin （HE） staining was conducted for the observation of SW620 cell morphology， and annexin-V/propidium iodide double staining for the determination of apoptosis rate in SW620 cells. Hoechst33258 staining and MDC staining were used for the observation of apoptosis and autophagy， respectively. Western blotting was employed to determine the protein levels of B-cell lymphoma/leukemia-2（Bcl-2）-associated X protein （Bax）， aspartate proteolytic enzyme-9 （Caspase-9）， activated aspartate proteolytic enzyme-3 （cleaved Caspase-3）， microtubule-associated protein 1 light chain 3 （LC3）， the mammalian homolog of yeast Atg6 （Beclin-1）， p62， phosphorylated PI3K （p-PI3K）， phosphorylated Akt （p-Akt）， and phosphorylated mTOR （p-mTOR） in SW620 cells. ResultBA inhibited the activity of SW620， HT29， and HCT116 cells in a concentration- and time-dependent manner. The cells treated with BA for 48 h had lower viability than those treated for 24 h （P<0.05， P<0.01）. The half maximal inhibitory concentration (IC50） value of BA at the time point of 48 h was also lower than that at the time point of 24 h （P<0.01）， and that for SW620 cells was the minimum. BA induced the apoptosis in a concentration-dependent manner and increased the autophagosomes. Compared with the blank group， BA increased the apoptosis rate （P<0.01）， up-regulated the protein levels of Bax， Caspase-9， cleaved Caspase-3， and LC3 Ⅱ （P<0.05， P<0.01）， and down-regulated the protein levels of p62， p-Akt， p-PI3K， and p-mTOR （P<0.01）. Additionally， medium- and high-dose BA up-regulated the protein level of beclin-1 （P<0.01）. ConclusionBA may inhibit the activity of SW620 cells by hindering the PI3K/Akt/mTOR signaling pathway to induce cell apoptosis and autophagy.

Explore the antiproliferative phytocompounds from ethanolic extracts of Citrus paradisi against liver cancer cell line by chemical analysis using TLC and FT-IR spectroscopy / Explorar os fitocompostos antiproliferativos de extratos etanólicos de Citrus paradisi contra linhagem de células de câncer de fígado por análise química usando espectroscopia de TLC e FT-IR

The aim of the present study was to evaluate the in vitro antiproliferative activity of ethanolic extract of leaves and fruits Citrus paradisi plant on HepG-2 liver cell lines by MTT (3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Hterazolium bromide) assay and to isolate and characterize the antiproliferative compounds by TLC (Thin layer chromatography) and FT-IR (Fourier transforms Infrared) spectroscopy. Qualitative phytochemical screening tests were performed to detect phytochemicals compounds from the crude extracts. Antioxidant activity of the plant extracts were characterized by using DPPH (2,2-Diphenyl-1-picrylhydrazyl) free radical scavenging method. The results showed that antioxidant activity using DPPH were found to be increased in a concentration dependent manner and decreased cell viability and cell growth inhibition in a dose dependent manner. The findings from this study indicated that fruit extract exhibited good antiproliferation and antioxidant potential. The seven functional groups of phytocompounds such as carboxylic acid, amine salt, aromatic compounds, cyclic alkene, aldehyde, fluoro compounds and alkene were detected by FT-IR which indicated that fruit extracts of Citrus paradisi possessed vast potential as a medicinal drug especially in liver cancer treatment.

O objetivo do presente estudo foi avaliar a atividade antiproliferativa in vitro do extrato etanólico de folhas e frutos da planta Citrus paradisi em linhagens de células hepáticas HepG-2 por MTT (3- (4, 5-dimetil-2-tiazolil) -2, Ensaio de brometo de 5-difenil-2H-terazólio) e isolar e caracterizar os compostos antiproliferativos por espectroscopia de TLC (cromatografia de camada fina) e FT-IR (infravermelho com transformadas de Fourier). Testes qualitativos de triagem fitoquímica foram realizados para detectar compostos fitoquímicos nos extratos brutos. A atividade antioxidante dos extratos vegetais foi caracterizada pelo método de eliminação de radicais livres DPPH (2,2-difenil-1-picrilhidrazil). Os resultados mostraram que a atividade antioxidante usando DPPH aumentou de uma maneira dependente da concentração e diminuiu a viabilidade celular e a inibição do crescimento celular de uma maneira dependente da dose. Os resultados deste estudo indicaram que o extrato de fruta exibiu bom potencial antiproliferação e antioxidante. Os sete grupos funcionais de fitocompostos, como ácido carboxílico, sal de amina, compostos aromáticos, alceno cíclico, aldeído, compostos de flúor e alceno, foram detectados por FT-IR, o que indicou que extratos de frutas de Citrus paradisi possuíam vasto potencial como medicamento, especialmente no tratamento de câncer do fígado.

Construction of HMGA2 knockout model of human papillary thyroid cancer cell line TPC-1 / 中华内分泌外科杂志

Objective:To construct a TPC-1 cell model that stably knocks out the HMGA2 by using CRISPR/Cas9 gene editing technology. Methods:Recombinant pLV[2gRNA]-EGFP:T2A:Puro- U6> {hHMGA2 [gRNA# A1]*}- U6>{hHMGA2 [gRNA#A2]*} of lentiviral plasmid vector was constructed: targeting HMGA2 Dual-gRNA sequence was designed, the synthesized Dual-gRNA fragment into pLV [2gRNA]-EGFP was cloned: T2A:Puro-U6 vector, extract a single clone for sequencing verification. the constructed recombinant plasmid vector with lentivirus was packed, and TPC-1 cells were infected, puromycin was used to obtain HMGA2 knock-out single clone, PCR and sequencing verification were performed, and real-time fluorescent quantitative qPCR was used to detect HMGA2 mRNA in cells Knockout efficiency. Results:After sequencing verification, pLV [2gRNA]-EGFP targeting HMGA2: T2A: Puro-U6>{hHMGA2 [gRNA#A1]*}-U6>{hHMGA2 [gRNA #A2]*} plasmid was successfully constructed; A single clone was picked for PCR identification and gene sequencing, TPC-1 cells were successfully obtained with HMGA2 gene completely knocked out; TPC-1 cells with HMGA2 knocked out were detected by real-time fluorescent quantitative qPCR, and they did not express HMGA2 mRNA.Conclusion:CRISPR/Cas9 gene editing technology enables us to construct a human papillary thyroid cancer cell line TPC-1 cell model with stable knockout of HMGA2.

Syringic acid induces cancer cell death in the presence of Cu (II) ions via pro-oxidant activity

Objective: To investigate the effects of syringic acid on HEK 293 and HepG2 cells in the absence and presence of exogenous Cu (II) and Fe (II) ions. Methods: The antiproliferative effects of syringic acid on HEK 293 and HepG2 cells in the absence and presence of exogenous Cu (II) and Fe (II) ions were examined by MTT assay. Additionally, colony-forming, reactive oxidative species (ROS) generation, apoptosis induction, autophagy, mitochondrial membrane potential, and mitochondrial mass were investigated. Results: At 24 and 72 h, no significant differences were observed in the viability of HepG2 cells between the control and syringic acid + Fe (II) groups. However, exposure of HepG2 cells to syringic acid + Cu (II) for 72 h reduced the cell viability significantly. Furthermore, ROS formation, induction of apoptosis, and autophagic vacuoles were significantly increased in HepG2 cells without marked changes in mitochondrial membrane potential and mitochondrial mass. Moreover, syringic acid + Cu (II) reduced the plating efficiency and surviving fraction significantly. Conclusions: The combination of syringic acid with Cu (II) was toxic to cancer cells and showed pro-oxidant activity. In addition, this combination induced autophagy in cancer cells with less cytotoxic effects on normal cells, which is a potential candidate for the development of novel therapeutics towards cancer.

Immune Checkpoint Molecules in Cancer Cells: Not in Their Places but on Their Duty / 中国生物化学与分子生物学报

Immune checkpoints represent a group of inhibitory receptor molecules that are expresses in the surface of immune cells and play a pivotal role in maintaining immune homeostasis. In recent years, several key immune checkpoint molecules such as CTLA-4 and PD-1, has been found to exist in some kinds of tumor cells. These ectopic expressed checkpoint molecules are named as “cancer cell-intrinsic immune checkpoint molecules”. Although our understanding on cancer cell-intrinsic immune checkpoint molecules is quite limited, emerging evidence suggests that the expression and the biological functions of such molecules in different types of cancer cells are heterozygous and diverse. In particular, the discovery of “adaptive immune-independent” regulation on cancer cell behaviors would potentially benefit to design a customized cancer immune therapy, as well as to develop new therapeutic strategies for cancer. In this review, we will briefly describe the timeline of the studies, deeply discuss the complicated biological functions and the regulatory mechanisms (CTLA-4 and PD-1 as representative examples). And finally we put forward a research perspective on cancer cell-intrinsic immune checkpoint molecules. This review aims to present and promote the studies of cancer cell-intrinsic immune checkpoint molecules to the broad scientific community.