OBJECTIVE: To explore the regulatory mechanism of human hepatocyte apoptosis induced by lysosomal membrane protein Sidt2 knockout. METHODS: The Sidt2 knockout (Sidt2^-/-) cell model was constructed in human hepatocyte HL7702 cells using Crispr-Cas9 technology.The protein levels of Sidt2 and key autophagy proteins LC3-II/I and P62 in the cell model were detected using Western blotting, and the formation of autophagosomes was observed with MDC staining.EdU incorporation assay and flow cytometry were performed to observe the effect of Sidt2 knockout on cell proliferation and apoptosis.The effect of chloroquine at the saturating concentration on autophagic flux, proliferation and apoptosis of Sidt2 knockout cells were observed. RESULTS: Sidt2^-/- HL7702 cells were successfully constructed.Sidt2 knockout significantly inhibited the proliferation and increased apoptosis of the cells, causing also increased protein expressions of LC3-II/I and P62(P < 0.05) and increased number of autophagosomes.Autophagy of the cells reached a saturated state following treatment with 50 μmol/L chloroquine, and at this concentration, chloroquine significantly increased the expressions of LC3B and P62 in Sidt2^-/- HL7702 cells. CONCLUSION Sidt2 gene knockout causes dysregulation of the autophagy pathway and induces apoptosis of HL7702 cells, and the latter effect is not mediated by inhibiting the autophagy-lysosomal pathway.
Humans ; Lysosome-Associated Membrane Glycoproteins/metabolism* ; Autophagy ; Apoptosis ; Hepatocytes ; Lysosomes/metabolism* ; Chloroquine/pharmacology* ; Nucleotide Transport Proteins/metabolism*

OBJECTIVE: To explore the combined pro-apoptosis effect of HSP90 inhibitor BIIB021 and chloroquine (CQ) in chronic myeloid leukemia (CML) cells bearing T315I mutation and its mechanism. METHODS: The p210-T315I cells were divided into 4 groups by different treatment: control, BIIB021, CQ, and BIIB021 + CQ. After treated with BIIB021 or/and CQ for 24 hours, Annexin V/PI binding assay was used to detect apoptosis rates of CML cells. DAPI staining was used to observe nuclear fragmentation, and Western blot was used to detect the expression of caspase 3, PARP (apoptosis related proteins) and p62, LC3-I/II (autophagy related proteins). P210-T315I cells were inoculated subcutaneously into mice and CML mouse models were established. The mice in treatment groups were injected with BIIB021 and/or CQ while mice in control group were treated with PBS and normal saline. The tumor volume of mice was measured every 4 days, and protein level of cleaved-caspase 3 and LC3-II in tumor tissue were detected by immunohistochemistry. RESULTS: The results showed that BIIB021 induced apoptosis of CML cells in a dose-dependent manner ( r=0.91). CQ could enhance the apoptosis-inducing effect of BIIB021. Flow cytometry analysis results showed that the apoptosis rate of p210-T315I cells in combination group was higher than that in BIIB021 or CQ only group (P<0.05). DAPI staining showed nuclear fragmentation in combination group could be observed more obviously. Western blot analysis showed that BIIB021 could induce LC3-I to convert to LC3-II and decrease p62 protein levels (P<0.05). Moreover, the combination group had higher expression of LC3-II, p62 (P<0.05), activated PARP and activated caspase 3 than BIIB021 only group (P<0.05). Besides, experiment in vivo showed the mean tumor volume in co-treatment group was lower than that in single drug group (P<0.01). Immunohistochemistry of tumor tissue also showed the protein level of cleaved-caspase 3 and LC3-II in combined group was higher than that in BIIB021 only group. CONCLUSION HSP90 inhibitor BIIB021 induced significant apoptosis of CML cells bearing T315I both in vivo and in vitro. CQ can enhance this effect probably by autophagy inhibition.
Adenine/analogs & derivatives* ; Animals ; Apoptosis ; Autophagy ; Caspase 3/metabolism* ; Cell Line, Tumor ; Chloroquine/therapeutic use* ; Fusion Proteins, bcr-abl/pharmacology* ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy* ; Mice ; Mutation ; Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use* ; Pyridines

Animals ; Chloroquine/pharmacology* ; Longevity ; Rats

OBJECTIVE: To determine whether chloroquine (CQ), an often used inhibitor of late autophagy and autophagosome/lyosome fusion, can inhibit proliferation of renal carcinoma cells and investigate its effect on sunitinib (ST)-induced apoptosis. METHODS: Renal carcinoma cell line 786 O and ACHN had been used as cellular model and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay was carried out to detect the cell viability in response to CQ or ST treatment. Both transmission electron microscope and immunoblotting had been employed to observe apoptotic and autophagic process. To examine the involvement of autophagy in ST-dependent apoptosis, autophagy had been inhibited either chemically or genetically via utilizing autophagy inhibitor or specific small interference RNA (siRNA) targeted to either Ulk1 (unc-51-like kinase 1) or LC3 (microtubule associated protein 1 light chain 3 fusion protein), two essential autophagic proteins. RESULTS: Both ST and CQ induced cell viability loss, indicating that either of them could inhibit renal cancer cell proliferation. Clone formation experiments confirmed the aforementioned results. Furthermore, the combined ST with CQ synergistically promoted the loss of cell viability. By transmission electron microscopy and immunoblotting, we found that the ST induced both autophagy and caspase-dependent apoptosis. While 3-MA, an early autophagy inhibitor, reduced the ST-induced cleavage of poly (ADP-ribose) polymerase-1 (PARP-1), a substrate of caspase 3/7 and often used marker of caspase-dependent apoptosis, CQ promoted the ST-dependent PARP-1 cleavage, indicating that the early and late autophagy functioned differentially on the ST-activated apoptotic process. Moreover, the knock down of either Ulk1 or LC3 decreased the ST-caused apoptosis.Interestingly, we observed that rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin) and an inducer of autophagy, also showed to inhibit cell viability and increased the cleavage of PARP-1 in the ST-treated cells, suggesting that autophagy was likely to play a dual role in the regulation of the ST-induced apoptosis. CONCLUSION ST activates both apoptotic and autophagic process in renal carcinoma cells. Although autophagy precedes the ST-induced apoptosis, however, early and late autophagy functions differentially on the apoptotic process induced by this compound. Additionally, ST can coordinate with the inducer of autophagy to inhibit the cell proliferation. Further research in this direction will let us illuminate to utilize CQ as a potential drug in the treatment of renal carcinoma.
Animals ; Antineoplastic Agents/pharmacology* ; Antirheumatic Agents/pharmacology* ; Apoptosis/drug effects* ; Autophagy/drug effects* ; Caspases ; Cell Line, Tumor ; Chloroquine/pharmacology* ; Kidney Neoplasms/drug therapy* ; Sunitinib/pharmacology*

OBJECTIVES: To investigate the role of autophagy inhibitor chloroquine (CQ) in acute ethanol-induced liver injury and its mechenism. METHODS: Twenty-one C57BL/6 male mice were randomly divided into three groups:control group, ethanol group, CQ + ethanol group (=7). Mice in ethanol group were administered 33% (v/v) ethanol at a dose of 4.5 g/kg body weight. Ethanol-induced liver steatosis in each group was detected by hematoxylin and eosin staining. Hepatic lipid accumulation was detected by staining with Oil red O. Hepatic tissue triglyceride (TG) levels, serum aspartate aminotransferase(AST) and alanine aminotransferase(ALT) were determined by biochemical assays. Protein expression of microtubule-associated protein 1 light chain 3(LC3) and nuclear factorκB p65(NF-κB p65) were measured by Western blot and immunofluorescence. Pro-inflammatory factors tumor necrosis factor-α(TNF-α)、interleukin 6(IL-6) were detected by ELISA. RESULTS: Compared with control group, ethanol induced liver injury proved by accumulation of hepatic lipids, TG levels, AST and ALT activities were significantly increased by ethanol, protein expression of LC3-Ⅱ was also markedly increased by ethanol. Compared with ethanol group, addition of CQ increased furtherthe level of LC3-Ⅱexpression, and TG amount, serum AST and ALT activities, and the expression of NF-κB p65, TNF-αand IL-6. CONCLUSIONS Acute ethanol-intake could induce liver steatosis and inflammation, and autophagy inhibitor CQ exacerbatedethanol-induced liver injury, suggested that autophagy might be protective effect in acute ethanol-induced liver disease.
Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Autophagy ; drug effects ; Chloroquine ; pharmacology ; Interleukin-6 ; analysis ; Liver ; drug effects ; Liver Diseases, Alcoholic ; drug therapy ; Male ; Mice ; Mice, Inbred C57BL ; Microtubule-Associated Proteins ; metabolism ; Random Allocation ; Transcription Factor RelA ; metabolism ; Triglycerides ; analysis ; Tumor Necrosis Factor-alpha ; analysis

Chloroquine ; pharmacology ; Down-Regulation ; Gene Expression ; drug effects ; HEK293 Cells ; Humans ; Leupeptins ; pharmacology ; Membrane Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Microscopy, Fluorescence ; Protein Binding ; Protein Subunits ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Shab Potassium Channels ; genetics ; metabolism

OBJECTIVETo investigate the effect of chloroquine on airway hyperresponsiveness in asthmatic mice and explore the possible mechanism.

METHODSBalb/c mouse models of asthma established using OVA received intraperitoneal injections of chloroquine, dexamethasone, or both prior to OVA challenge. Within 24 h after the final challenge, airway hyper- responsiveness (AHR) of the mice was assessed, and the total cell count and the counts of different cell populations in the bronchoalveolar lavage fluid (BALF) were determined under light microscopy. The severity of lung inflammation was evaluated using HE staining, and the concentrations of IL-6 and PGF2α in the BALF were detected by enzyme-linked immunosorbent assay (ELISA).

RESULTSChloroquine pretreatment significantly decreased AHR (P<0.001) in the asthmatic mice and reduced the total cell count (P<0.01), eosinophils (P<0.001), neutrophils (P<0.01), and PGF2α levels in the BALF. Chloroquine combined with low-dose dexamethasone significantly lessened inflammations around the bronchioles (P<0.05) and blood vessels (P<0.01) in the lung tissue, and obviously lowered IL-6 (P<0.05) and PGF2α (P<0.001) in the BALF in the asthmatic mice.

CONCLUSIONChloroquine can inhibit AHR in asthmatic mice and produce better anti-inflammatory effect when combined with dexamethasone for treatment of neutrophilic asthma.

Animals ; Asthma ; chemically induced ; drug therapy ; Bronchoalveolar Lavage Fluid ; cytology ; Chloroquine ; pharmacology ; Dexamethasone ; pharmacology ; Dinoprost ; metabolism ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Eosinophils ; cytology ; Inflammation ; pathology ; Interleukin-6 ; metabolism ; Leukocyte Count ; Lung ; pathology ; Mice ; Mice, Inbred BALB C ; Neutrophils ; cytology

Recent data have revealed that inhibiting autophagy exacerbates lipid accumulation in hepatocytes and nitrite treatment reduces total triglyceride levels in the high-fat diet mice. Therefore, the present study aimed to determine the effects of nitrite on simple hepatic steatosis and the possible role of autophagy. Firstly, steatotic L-02 cells were induced by incubating L-02 cells with 1.2 mmol · L(-1) oleic acid (OA) for 24 h. Secondly, steatotic L-02 cells were treated with 0.2 mmol · L(-1) sodium nitrite (SN) plus 3-methyladenine (3-MA), or chloroquine (CQ) for 24 h, and then lipid accumulation was measured with oil red O staining and triglyceride quantification. The notable steatosis could be observed in L-02 cells following exposure to 1.2 mmol · L(-1) OA for 24 h. Treatment with 0.2 mmol · L(-1) sodium nitrite reduced lipid accumulation in steatotic L-02 cells. 3-MA weakened the ability of sodium nitrite to ameliorate hepatic steatosis. Additionally, the sodium nitrite increased number of LC3-II immunostaining puncta and LC3-II protein expression was confirmed by immunofluorescence or Western blot analysis, and the effects were enhanced by CQ treatment. The number of increased cytoplasm vacuoles and lysosomes increased was confirmed by phase contrast and fluorescence microscope respectively. The increased autolysosome was detected by electron microscopy, this phenomenon could be reversed by CQ treatment. These data demonstrated that sodium nitrite enhanced the autophagic flux and decomposition of triglycerides in steatotic L-02 cells.
Adenine ; analogs & derivatives ; Autophagy ; Blotting, Western ; Cells, Cultured ; Chloroquine ; Cytoplasm ; Fatty Liver ; Hepatocytes ; drug effects ; Humans ; Lipid Metabolism ; drug effects ; Microscopy, Fluorescence ; Microtubule-Associated Proteins ; metabolism ; Oleic Acid ; Sodium Nitrite ; pharmacology ; Triglycerides

OBJECTIVETo investigate the effect of chloroquine in reversing multidrug resistance (MDR) of HNE1/DDP cell line and explore the mechanism.

METHODSMTT assay was used to detect the cell viability of HNE1 and HNE1/DDP after exposure to different concentrations of DDP (2, 4, 8, 16 and 32 µmol/L) and different concentrations of chloroquine (5, 10, 20, 40, and 80 µmol/L). q-PCR was used to assess the expression of MDR1 mRNA and Western blotting was employed to detect P-glycoprotein (P-gp) expression in HNE1 and HNE1/DDP cells exposed to 5 and 10 µmol/L chloroquine. The cell apoptosis rate of HNE1 and HNE1/DDP cells exposed to 10 and 20 µmolchloroquine was determined by PI assay.

RESULTSChloroquine exposure caused dose-dependent suppression of the proliferation in both HNE1 and HNE1chloroquine.

CONCLUSIONChloroquine can reverse multidrug resistance in HNE1
ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Cell Line, Tumor ; Chloroquine ; pharmacology ; Down-Regulation ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Humans

OBJECTIVETo understand the druggability of the bioactive compounds from traditional herbal formulations "Nilavembu Kudineer" and "Swasthya Raksha Amruta Peya" to heal chikungunya virus (CHIKV) infection.

METHODSThe efficiency of twenty novel chemical entities from "Nilavembu Kudineer" and "Swasthya Raksha Amruta Peya" to inhibit CHIKV infection in silico were evaluated. Ligands were prepared using Ligprep module of Schrödinger. Active site was identified using SiteMap program. Grid box was generated using receptor grid generation wizard. Molecular docking was carried out using Grid Based Ligand Docking with Energetics (GLIDE) program.

RESULTSMolecular docking studies showed that among twenty compounds, andrographoside, deoxyandrographoside, neoandrographolide, 14-deoxy-11-oxoandrographolide, butoxone and oleanolic acid showed GLIDE extra precision (XP) score of -9.10, -8.72, -8.25, -7.38, -7.28 and -7.01, respectively which were greater than or comparable with chloroquine (reference compound) XP score (-7.08) and were found to interact with the key residues GLU 1043, LYS 1045, GLY 1176, LEU 1203, HIS 1222 and LYS 1239 which were characteristic functional unit crucial for replication of CHIKV.

CONCLUSIONThe binding affinity and the binding mode of chemical entities taken from herbal formulations with non-structural protein 2 protease were understood and our study provided a novel strategy in the development and design of drugs for CHIKV infection.

Antiviral Agents ; chemistry ; pharmacology ; Catalytic Domain ; Chikungunya virus ; drug effects ; enzymology ; Chloroquine ; chemistry ; pharmacology ; Complementary Therapies ; Cysteine Endopeptidases ; chemistry ; Drug Design ; Hydrogen Bonding ; Ligands ; Molecular Docking Simulation ; Phytochemicals ; chemistry ; Protein Structure, Secondary