This study aims to delve into the influences and underlying mechanisms of Banxia Xiexin Decoction(BXD) on the proliferation, apoptosis, invasion, and migration of colon cancer cells. Firstly, the components of BXD in blood were identified by UPLC-MS/MS, and subsequently the content of these components were determined by HPLC. Then, different concentrations of BXD were used to treat both the normal intestinal epithelial cells(NCM460) and the colon cancer cells(HT29 and HCT116). The cell viability and apoptosis were examined by the cell counting kit-8(CCK-8) and flow cytometry, respectively. Western blot was employed to determine the expression of the apoptosis regulators B-cell lymphoma-2(Bcl-2) and Bcl-2-associated X(Bax). The cell wound healing assay and Transwell assay were employed to measure the cell migration and invasion, respectively. Additionally, Western blot was employed to determine the expression levels of epithelial-mesenchymal transition(EMT)-associated proteins, including epithelial cadherin(E-cadherin), neural cadherin(N-cadherin), and vimentin. The protein and mRNA levels of the factors in the poly(ADP-ribose) glycohydrolase(PARG)/poly(ADP-ribose) polymerase 1(PARP1)/nuclear factor kappa-B p65(NF-κB p65) signaling pathway were determined by Western blot and RT-qPCR, respectively. The results demonstrated that following BXD intervention, the proliferation of HT29 and HCT116 cells was significantly reduced. Furthermore, BXD promoted the apoptosis, enhanced the expression of Bcl-2, and suppressed the expression of Bax in colon cancer cells. At the same time, BXD suppressed the cell migration and invasion and augmented the expression of E-cadherin while diminishing the expression of N-cadherin and vimentin. In addition, BXD down-regulated the protein and mRNA levels of PARG, PARP1, and NF-κB p65. In conclusion, BXD may inhibit the malignant phenotypes of colon cancer cells by mediating the PARG/PARP1/NF-κB signaling pathway.
Colonic Neoplasms/pathology* ; Drugs, Chinese Herbal/pharmacology* ; Phenotype ; Signal Transduction/drug effects* ; Cell Proliferation/drug effects* ; Apoptosis ; Cell Movement/drug effects* ; Neoplasm Invasiveness ; HCT116 Cells ; Proto-Oncogene Proteins c-bcl-2/biosynthesis* ; Humans ; Poly (ADP-Ribose) Polymerase-1 ; Glycoside Hydrolases ; bcl-2-Associated X Protein ; NF-kappa B p50 Subunit

This study aims to investigate the effect of ethoxysanguinarine(Eth) on cisplatin(DDP)-resistant human gastric cancer cells and decipher the underlying mechanism. The human gastric cancer cell line SGC7901 and the DDP-resistant cell line SGC7901/DDP were used as the cell models. Western blot was employed to determine the expression levels of multidrug resistance-related proteins, and methyl thiazolyl tetrazolium(MTT) assay to detect the proliferation of SGC7901 and SGC7901/DDP cells exposed to DDP. After treatment with different concentrations of Eth, the proliferation of SGC7901 and SGC7901/DDP cells was detected by MTT assay, trypan blue exclusion assay, colony formation assay, and high-content imaging and analysis system. The apoptosis of SGC7901/DDP cells was detected by flow cytometry with Annexin V-FITC/PI staining. GFP-LC3 transfection was carried out to detect the effect of Eth on the autophagy of SGC7901/DDP cells. The expression levels of the multidrug resistance-related protein P-glycoprotein(P-gp), the apoptosis-related proteins [caspase-9, caspase-3, and poly(ADP-ribose) polymerase(PARP)], the autophagy-related protein light chain 3-Ⅱ(LC3-Ⅱ), the key effectors [mammalian target of rapamycin(mTOR), 70 kDa ribosomal protein S6 kinase(P70 S6 K), and 4 E binding protein 1(4 E-BP1)] of the mammalian target of rapamycin complex 1(mTORC1) signaling pathway, cancerous inhibitor of protein phosphatase 2A(CIP2A), and protein kinase B(Akt) were measured by Western blot. The mRNA level of CIP2A in the SGC7901/DDP cells exposed to Eth for 24 h was analyzed by RT-qPCR. After SGC7901/DDP cells were transfected with CIP2A expression vector pcDNA3.1-HA-CIP2A and treated with different concentrations of Eth, MTT assay was used to determine the prolife-ration of SGC7901/DDP cells and Western blot to detect the expression levels of related proteins. The interaction sites of Eth and CIP2A were predicted by molecular docking. The affinity between Eth and CIP2A was determined by drug affinity responsive target stability(DARTS) assay. The pharmacokinetic properties and drug-like activity of Eth were predicted by SwissADME. The results indicated that SGC7901/DDP cells were more sensitive to Eth than SGC7901 cells. Eth significantly inhibited proliferation and colony formation and changed the morphology, roundness, and area of SGC7901/DDP cells. Eth treatment caused the nucleus shrinking and significantly increased the apoptosis rate of the cells. Furthermore, Eth down-regulated the expression of caspase-9 and caspase-3 precursors and promoted the cleavage of PARP, which suggested that Eth induced the apoptosis of SGC7901/DDP cells. The GFP-LC3 in Eth-treated cells showed speckled aggregation. The up-regulated expression of LC3-Ⅱ by Eth indicated that Eth activated the autophagy of SGC7901/DDP cells. Eth down-regulated the expression of P-gp, the phosphorylation of mTOR, P70 S6K, and 4E-BP1, the expression of CIP2A, and the phosphorylation of Akt. Additionally, it increased the activity of PP2A, and had no significant effect on the expression of CIP2A in SGC7901/DDP cells. CIP2A overexpression antagonized the inhibition of cell proliferation and the activation of autophagy by Eth. Molecular docking suggested that Eth bound to CIP2A. The results of DARTS assay further proved the above binding effect. Eth has potential drug-like activity. The above results demonstrated that Eth inhibited the proliferation, induced the apoptosis, and activated the autophagy of SGC7901/DDP cells by targeting CIP2A and then down-regulating PP2A/mTORC1 signaling pathway. This study provided a new target for the treatment of cisplatin-resistant gastric cancer.
Humans ; Cisplatin/therapeutic use* ; Caspase 9/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Caspase 3/metabolism* ; Stomach Neoplasms/metabolism* ; Drug Resistance, Neoplasm ; Antineoplastic Agents/therapeutic use* ; Molecular Docking Simulation ; Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use* ; Autophagy ; Apoptosis ; Cell Proliferation ; Apoptosis Regulatory Proteins/metabolism* ; Mechanistic Target of Rapamycin Complex 1/metabolism* ; Cell Line, Tumor

OBJECTIVE: To investigate the effect of dihydroartemisinin (DHA) combined with arsenic trioxide (ATO) on the viability and apoptosis of acute myeloid leukemia (AML) FLT3-ITD mutant cell line MOLM13 and its mechanism. METHODS: MOLM13 cells were treated with DHA or ATO alone or in combination. The viability of MOLM13 cells was detected by CCK-8 assay, cell proliferation was observed by colony formation assay, cell apoptosis and reactive oxygen species (ROS) level were measured by flow cytometry, and the expression levels of proteins related to apoptosis were detected by Western blot. RESULTS: Compared with the control group, treatment with DHA and ATO alone or in combination could inhibit cell proliferation, activate ROS formation, and finally induce cell apoptosis. DHA in combination with ATO produced a synergistic effect. Western blot analysis showed that DHA combined with ATO could significantly upregulate the level of c-PARP and activate apoptosis via inhibition of Mcl-1 and FLT3-ITD. CONCLUSION DHA combined with ATO induces the apoptosis of FLT3-ITD AML cell line MOLM13 by inhibiting Mcl-1 pathway and activating FLT3-ITD protein degradation.
Apoptosis ; Arsenic Trioxide/therapeutic use* ; Artemisinins/therapeutic use* ; Cell Line, Tumor ; Humans ; Leukemia, Myeloid, Acute/drug therapy* ; Myeloid Cell Leukemia Sequence 1 Protein ; Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use* ; Reactive Oxygen Species/therapeutic use* ; Sincalide/therapeutic use* ; fms-Like Tyrosine Kinase 3

OBJECTIVE: "Multi-targeting" drugs can prove fruitful to combat drug-resistance of multifactorial disease-cervical cancer. This study envisioned to reveal if Thuja homeopathic mother tincture (MT) and its bioactive component could combat human papillomavirus (HPV)-16-infected SiHa cervical cancer cells since it is globally acclaimed for HPV-mediated warts. METHODS: Thuja MT was studied for its antiproliferative and antimigratory properties in SiHa cells followed by microscopic determination of reactive oxygen species (ROS) generation by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining and loss in mitochondrial membrane potential (MtMP) by rhodamine 123 (Rh123) staining. Apoptosis and autophagy inductions were studied by acridine orange/ethidium bromide (AO/EB) staining and immunoblot analyses of marker proteins. The bioactive component of Thuja MT detected by gas chromatography-mass spectrometry was studied for antiproliferative and antimigratory properties along with in silico prediction of its cellular targets by molecular docking and oral drug forming competency. RESULTS: Thuja MT showed significant antiproliferative and antimigratory potential in SiHa cells at a 50% inhibitory concentration (IC₅₀) of 17.3 µL/mL. An increase in DCFDA fluorescence and loss in Rh123 fluorescence prove that Thuja MT acted through the burst of ROS and loss in MtMP respectively. AO/EB-stained cells under the microscope and immunoblot analyses supported Thuja-induced cellular demise via dual pathways-apoptosis and autophagy. Immunoblots showed cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) along with upregulation of Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B)-II, and p62 proteins. Hence, the apoptotic cascade followed a caspase-3-dependent pathway supported by PARP-1 cleavage, while autophagic death was Beclin-1-dependent and mediated by accumulation of LC3BII and p62 proteins. Thujone, detected as the bioactive principle of Thuja MT, showed greater anti-proliferative and anti-migratory potential at an IC₅₀ of 77 µg/mL, along with excellent oral drug competency with the ability for gastrointestinal absorption and blood-brain-barrier permeation with nil toxicity. Molecular docking depicted thujone with the strongest affinity for mammalian target of rapamycin, phosphoinositide 3-kinase, and protein kinase B followed by B-cell lymphoma 2, murine double minute 2 and adenosine monophosphate-activated protein kinase, which might act as upstream triggers of apoptotic-autophagic crosstalk. CONCLUSION Robust "multi-targeting" anticancer potential of Thuja drug and thujone for HPV-infected cervical cancer ascertained its therapeutic efficacy for HPV infections.
Animals ; Apoptosis ; Autophagy ; Beclin-1/pharmacology* ; Bicyclic Monoterpenes ; Caspase 3 ; Cell Line, Tumor ; Female ; Humans ; Mammals/metabolism* ; Mice ; Molecular Docking Simulation ; Papillomavirus Infections/drug therapy* ; Phosphatidylinositol 3-Kinases ; Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use* ; Reactive Oxygen Species/metabolism* ; Thuja/metabolism* ; Uterine Cervical Neoplasms/pathology*

OBJECTIVE: To investigate the regulatory effect and mechanism of DNA methyltransferase 3A (DNMT3a) in hydroquinone-induced hematopoietic stem cell toxicity. METHODS: Cells (HSPC-1) were divided into 4 groups, that is A: normal HSPC-1; B: HQ-intervented HSPC-1; C: group B + pcDNA3 empty vector; D: group B + pcDNA3- DNMT3a. RT-qPCR and Western blot were used to detect the expression levels of DNMT3a and PARP-1 mRNA and protein, respectively. Cell morphology was observe; Cell viability and apoptosis rate of HSPC-1 were detected by MTT and flow cytometry, respectively. RESULTS: Compared with group A, the expression levels of DNMT3a mRNA and protein in HSPC-1 of group B were decreased, while PARP-1 mRNA and protein were increased (P<0.05); there was no significant difference in the above indexes between group C and group B; compared with group B, the expression levels of DNMT3a mRNA and protein showed increased, while PARP-1 mRNA and protein were decreased significantly in cells of group D transfected with DNMT3a (P<0.05). Cells in each group were transfected with DNMT3a and cultured for 24 h, HSPC-1 in group A showed high density growth and mononuclear fusion growth, while the number of HSPC-1 in group B and C decreased and grew slowly. Compared with group B and C, the cell growth rate of group D was accelerated. The MTT analysis showed that cell viability of HSPC-1 in group B were lower than that of group A at 24 h, 48 h and 72 h (P<0.05); after transfected with DNMT3a, the cell viability of HSPC-1 in group D were higher than that of group B at 24 h, 48 h and 72 h (P<0.05). The apoptosis rate of cells in group B was significantly higher than that of group A (P<0.001), while the apoptosis rate in group D was lower than that of group B (P<0.001). CONCLUSION DNMT3a may be involved in the damage of hematopoietic stem cells induced by hydroquinone, which may be related to the regulation of PARP-1 activity by hydroquinone-inhibited DNMT3a.
Apoptosis ; Cell Proliferation ; DNA Methyltransferase 3A ; Hematopoietic Stem Cells/drug effects* ; Humans ; Hydroquinones/toxicity* ; Poly (ADP-Ribose) Polymerase-1 ; RNA, Messenger/metabolism*

To investigate the inhibitory effects and mechanism of Cistanche tubulosa ethanol extract( CTEE) against oxygen-glucose deprivation/reperfusion( OGD/R)-induced PC12 cells neuronal injury. In this study,OGD/R-induced PC12 cells were used to explore the neuroprotective effects of CTEE( 12. 5,25,50 mg·L-1) by detecting cell viability with MTT assay,apoptosis with AO/EB and Hoechst 33258,mitochondrial membrane potential changes with JC-1 staining,mitochondrial oxidative stress with MitoSOX staining,as well as the apoptosis-related protein expression( PARP,cleaved PARP,caspase-3,cleaved caspase-3,Bax,Bcl-2) with Western blot. RESULTS:: showed that CTEE effectively protected OGD/R-induced neuronal injury and increased the survival rate of PC12 cells.AO/EB and Hoechst 33258 staining showed that CTEE could effectively inhibit apoptosis. Moreover,JC-1 and MitoSOX staining results showed that CTEE decreased mitochondrial stress and mitochondrial membrane potential imbalance in PC12 cells in a concentration-dependent manner. Meanwhile,CTEE could obviously suppress the activation of key proteins in mitochondrial apoptosis pathway such as caspase-3 and PARP,and significantly inhibit the rise of Bax and down-regulation of Bcl-2. In conclusion,CTEE has obvious protective effects on OGD/R-induced PC12 cells neuronal injury,potentially via inhibiting mitochondrial oxidative stress and apoptosis-related signaling pathway.
Animals ; Apoptosis ; Caspase 3 ; metabolism ; Cistanche ; chemistry ; Ethanol ; Glucose ; Neuroprotective Agents ; pharmacology ; Oxidative Stress ; Oxygen ; PC12 Cells ; Plant Extracts ; pharmacology ; Poly (ADP-Ribose) Polymerase-1 ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; bcl-2-Associated X Protein ; metabolism

The underground cane of Schizocapsa plantaginea (Hance) has long been used by Chinese ethnic minority as a constituent of anti-cancer formulae. Saponins are abundant secondary metabolic products located in the underground cane of this plant. The potential therapeutic effects of total saponins isolated from Schizocapsa plantaginea (Hance) (SSPH) on human hepatocellular carcinoma (HCC) were tested in vitro in human liver cancer cell lines, SMMC-7721 and Bel-7404. Apoptosis and cell cycle arrest were determined using flow cytometry, caspase activation was determined by ELISA, and PARP, cleaved PARP, mitogen-activated protein kinase (MAPK) expression and phosphorylation were measured using Western blotting analysis. In vivo anti-HCC effects of SSPH were verified in nude mouse xenograft model. SSPH exerted markedly inhibitory effect on HCC cell proliferation in time- and concentration-dependent manner. Moreover, SSPH significantly induced apoptosis through caspase-dependent signaling and arrested cell cycle at G/M phase. These anti-proliferation effects of SSPH were associated with up-regulated phosphorylation of extracellular signal-regulated kinase-1/2 (Erk1/2) and c-jun-NH2-kinase-1/2 (JNK1/2) and reduced phosphorylation of p38MAPK. Furthermore, inhibitors of ERK, UO126, and JNK, SP600125 inhibited the anti-proliferation effects by SSPH, suggesting that Erk and JNK were the effector molecules in SSPH induced anti-proliferative action. During in vivo experiments, SSPH was found to inhibit xenograft tumor growth in nude mice, with a similar mechanism in vitro. Our study confirmed that SSPH exerted antagonistic effects on human liver cancer cells both in vitro and in vivo. Molecular mechanisms underlying SSPH action might be closely associated with MAPK signaling pathways. These results indicated that SSPH has potential therapeutic effects on HCC.
Animals ; Antineoplastic Agents ; isolation & purification ; pharmacology ; toxicity ; Apoptosis ; drug effects ; Caspases ; genetics ; metabolism ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Dioscoreaceae ; chemistry ; Heterografts ; drug effects ; growth & development ; Humans ; Inhibitory Concentration 50 ; Liver Neoplasms ; drug therapy ; metabolism ; pathology ; MAP Kinase Signaling System ; drug effects ; Mice ; Mice, Nude ; Phosphorylation ; drug effects ; Plant Tubers ; chemistry ; Poly (ADP-Ribose) Polymerase-1 ; metabolism ; Saponins ; isolation & purification ; pharmacology ; toxicity

The molecular mechanism of DNA damage induced by hydroquinone (HQ) remains unclear. Poly(ADP-ribose) polymerase-1 (PARP-1) usually works as a DNA damage sensor, and hence, it is possible that PARP-1 is involved in the DNA damage response induced by HQ. In TK6 cells treated with HQ, PARP activity as well as the expression of apoptosis antagonizing transcription factor (AATF), PARP-1, and phosphorylated H2AX (γ-H2AX) were maximum at 0.5 h, 6 h, 3 h, and 3 h, respectively. To explore the detailed mechanisms underlying the prompt DNA repair reaction, the above indicators were investigated in PARP-1-silenced cells. PARP activity and expression of AATF and PARP-1 decreased to 36%, 32%, and 33%, respectively, in the cells; however, γ-H2AX expression increased to 265%. Co-immunoprecipitation (co-IP) assays were employed to determine whether PARP-1 and AATF formed protein complexes. The interaction between these proteins together with the results from IP assays and confocal microscopy indicated that poly(ADP-ribosyl)ation (PARylation) regulated AATF expression. In conclusion, PARP-1 was involved in the DNA damage repair induced by HQ via increasing the accumulation of AATF through PARylation.
Antioxidants ; toxicity ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Cell Line ; DNA Damage ; drug effects ; Gene Expression Regulation ; drug effects ; Gene Silencing ; Histones ; genetics ; metabolism ; Humans ; Hydroquinones ; toxicity ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; genetics ; metabolism ; Protein Transport ; Repressor Proteins ; genetics ; metabolism

OBJECTIVETo explore whether MG-132 could enhance the anti-tumor activity of obatoclax against esophageal cancer cell line CaES-17.

METHODSMTT assay was used to determine the cytotoxicity of obatoclax and MG-132 in CaES-17 cells. The IC(50) of obatoclax and MG-132 were used to determine the molar ratio (1:2.4) of the two drugs for combined treatment of the cells. The concentrations of obatoclax and MG-132 ranged from 1/8 IC(50) to 4 IC(50) after serial dilution, and their combination index (CI) was calculated using CompuSyn software. The expression of ubiquitin and the cleavage of PARP, caspase-9, phospho-histone H3 and phospho-aurora A/B/C in the exposed cells were examined with Western blotting; the cell apoptosis was measured by flow cytometry with Annexin V staining, and the percentage of cells in each cell cycle phase was also determined by flow cytometry.

RESULTSThe CI of obatoclax and MG-132 was 0.296 for a 50% inhibition of Caes-17 cells and was 0.104 for a 95% inhibition. The cells treated with obatoclax or MG-132 alone showed increased expression of ubiquitin and cleavage of PARP and caspase-9. Compared with the cells treated with obatoclax or MG-132 alone, the cells with a combined treatment exhibited significantly increased expression of ubiquitin, cleavage of PARP and caspase-9, and expression of phospho-Histone H3 (P<0.05). The combined treatment of the cells also resulted in significantly increased expression of phospho-Aurora A/B/C compared with obatoclax treatment alone. The cells with the combined treatment showed significantly higher percentages of apoptotic cells and cells in sub-G(1) and G(2)/M phases compared with the cells treated with either of the drugs (P<0.05).

CONCLUSIONObatoclax combined with MG-132 shows a significant synergistic anti-tumor effect against esophageal cancer CaES-17 cells by inducing apoptosis and cell cycle arrest.

Apoptosis ; Caspase 9 ; metabolism ; Cell Cycle Checkpoints ; Cell Line, Tumor ; drug effects ; Esophageal Neoplasms ; pathology ; Histones ; metabolism ; Humans ; Leupeptins ; pharmacology ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism ; Pyrroles ; pharmacology

OBJECTIVETo investigate the effects of telocinobufagin on viability and apoptosis of colorectal cancer (CRC) cells and explore the mechanism of telocinobufagin-induced apoptosis.

METHODSMTT assay was performed to detect the viability of CRC cells exposed to telocinobufagin. Nuclear staining with Hoechst 33342 and flow cytometry were used to analyze the cell death of CRC cells. Expressions of proteins related with cell apoptosis and oxidative stress were determined with Western blotting.

RESULTSTelocinobufagin decreased the viability of CRC cells in a time- and dose-dependent manner. The presence of karyopycnosis and apoptotic bodies together with the results of flow cytometry suggested that telocinobufagin induced cell apoptosis to cause cell death. Western blotting showed that telocinobufagin exposure of the cells resulted in upregulated p53 and Bax protein expressions and promoted cleavage of caspase 9 and PARP. Telocinobufagin induced phosphorylation of Bad and PARP cleavage, and suppressed phosphorylation of IKBα and TAK1 and expression of survivin in the cells.

CONCLUSIONTelocinobufagin can decrease the viability of CRC cells by inducing cell apoptosis, which involves p53-mediated Bax activation and inhibition of the IAP pathway.

Apoptosis ; Bufanolides ; pharmacology ; Caspase 9 ; metabolism ; Cell Survival ; Colorectal Neoplasms ; pathology ; Humans ; MAP Kinase Kinase Kinases ; metabolism ; NF-KappaB Inhibitor alpha ; metabolism ; Oxidative Stress ; Poly (ADP-Ribose) Polymerase-1 ; metabolism ; Tumor Cells, Cultured ; Tumor Suppressor Protein p53 ; metabolism ; bcl-2-Associated X Protein ; metabolism ; bcl-Associated Death Protein ; metabolism