Artesunate inhibits development of esophageal squamous cell carcinoma via regulating expression of Skp2 and P21北大核心CSCD

Aim To clarify the regulatory effect of Artesunate(ART) on tumor cell function and cell cycle in the pathological process of esophageal squamous cell carcinoma(ESCC). Methods KYSE450 and TE14 cells were treated with different concentrations of ART. The cells treated with 0 mg •L-1 ART were used as the control group, and the cells treated with 30 mg•L -1ART were used as the experimental group. MTT assay and cloning formation experiment were used to sense cell growth. Transwell assay was applied to test the invasion and migration capacity of ART treated group and blank group separately,flow cytometry was employed to examine c ell cycle. The Mechanism of Artesunate on Escc by RNA-Seq was detected. Differential Genes We value USIDANG RT-QPCR. Results Compared with the Control Group, The P Roliferation, Invasion and Migration of Escc Cells Were SUPPRSSED CONSIDERABLY AFTER Art Treatment (P <0.01). According to the analysis of sequencing results, ART might inhibit the occurrence and development of tumor by affecting the cell cycle. After ART treatment, the ratio of KYSE450 cells at G1 phase boosted 18.02%. The proportion of KYSE450 cells in G1 phase increased significantly in the back of adding ART(24.12%±2.62% vs 42.14%±0.65%, P<0.01), and the percentage of TE14 cells at G 1phase also increased significantly after ART therapy(36.25%±0.21% vs 42.19%±0.38%, P<0.01). Skp2, a gene closely related to cell cycle regulation, decreased and its downstream target gene P21 increased. Conclusions ART, a Chinese patent medicine, can inhibit the proliferation, invasion and migration of ESCC cells, accelerate apoptosis and cause cell arrest through regulating the expression of Skp2 and P21. This study takes the re-application of Chinese patent medicine which is used against malaria originall y as the innovation point to provide a reference basis for the development of new safety, effective and economical targeted drugs for ESCC. It also provides scientific ideas for the development of a new treatment scheme of radiotherapy combined with ESCC. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Notch1 signaling inhibits growth of EC109 esophageal carcinoma cells through downmodulation of HPV18 E6/E7 gene expression

Aim： To investigate the role of the Notch1 signaling pathway in growth arrest of an esophageal carcinoma cell line （EC109） in vitro and the mechanism involved. Methods： An intracellular domain of Notch1 （ICN） was transfected into cultured EC109 cells by lipofectamine transfection. Subsequently, the proliferation of the transfected cells was measured by an MTT assay. Cell cycle distribution was analyzed by flow cytometry. Human papillomavirus type 18 （HPV18） E6/E7 mRNA expression was detected by RT-PCR, and p53 protein expression was detected by Western blot. Results： Activation of Notch1 signaling resulted in inhibition of EC109 cell proliferation with the induction of G2/M arrest, downmodulation of HPV18 E6/E7 gene expression, and upregulation of p53 expression. Conclusion：Repression of HPV18 E6/E7 expression by Notch1 signaling results in the activation of p53-mediated pathways with concomitant growth suppression of HPV18-positive EC109 cells.     

Topotecan inhibits cancer cell migration by down-regulation of chemokine CC motif receptor 7 and matrix metalloproteinases

Aim： The aim of this study was to investigate the effect of topotecan （TPT） on cancer cell migration. Methods： Growth inhibition of TPT was analyzed by MTT assay, and cancer cell migration was measured by transwell double chamber assay. To verify the effect of TPT on the chemokine receptors CXCR4 and CCR7, quantitative PCR, semiquantitative PCR and Western blot analysis were performed. The secretion of MMP-2 and MMP-9 was detected by enzymelinked immunosorbent assay （ELISA） and gelatin zymography. To evaluate possible contributions of CCR7 to MMP secre- tion, the overexpression vectors pcDNA3.1^＋-CCR7 and CCR7 siRNA were transiently transfected into MDA-MB-435 cells. Results： TPT inhibited cancer cell migration in a dose-dependent manner. Additionally, TPT significantly decreased the expression of CCR7 in both MDA-MB-435 and MDA-MB-231 cells and moderately reduced the expression of CXCR4 in MDA-MB-435 cells. The secretion of MMPs （MMP-2, MMP-9） was also inhibited by TPT. Overexpression of CCR7 increased the secretion of MMP-2/9 and cancer cell migration, whereas knockdown of CCR7 reduced active MMP-2/9 production and migration of MDA-MB-435 cells. Conclusion： TPT inhibited cancer cell migration by down-regulation of CCR7 and MMPs （MMP-2 and MMP-9）.     

Overexpression of acetylcholinesterase inhibited cell proliferation and promoted apoptosis in NRK cells

AIM: To study the potential function of acetylcholinesterase (AChE) in apoptosis through overexpression of AChE in Normal Rat Kidney (NRK) cells. METHODS: AChE activity was detected by the method of Karnovsky and Roots. Activated caspase-3 was analyzed by Western blotting and immunoflurescence with antibody special to activated caspase-3 fragment. The expression plasmids were constructed in pcDNA3.1 containing AChE gene or a fragment of AChE antisense that were got from RT-PCR. Stable expression cell lines were selected by G418 in cells transfected by lipofection. AChE expression was analyzed by RT-PCR and Western blotting. The proliferation rates of transfected cells were examined by the growth curve and cloning efficiency. MTT assay was used to analyze the cell viability. RESULTS: The proliferation rate of the cells transfected with AChE was retarded and the cloning efficiency was lower (28.2 %±3.1 % and 48.7 %±2.1 %) than cells transfected with vector (56.1 %±0.3 %) or AChE-antisense (77.7 %±2.2 %     

Inhibition of epidermal growth factor receptor expression by RNA interference in A549 cells

AIM: To investigate the biological features of A549 cells in which epidermal growth factor (EGF) receptors expression were suppressed by RNA interference (RNAi). METHODS: A549 cells were transfected using short small interfering RNAs (siRNAs) formulated with Lipofectamine 2000. The EGF receptor numbers were determined by Western blotting and flowcytometry. The antiproliferative effects of sequence specific double stranded RNA (dsRNA) were assessed using cell count, colony assay and scratch assay. The chemosensitivity of transfected cells to cisplatin was measured by MTT. RESULTS: Sequence specific dsRNA-EGFR down-regulated EGF receptor expression dramatically. Compared with the control group, dsRNA-EGFR reduced the cell number by 85.0 %, decreased the colonies by 63.3 %, inhibited the migration by 87.2 %, and increased the sensitivity of A549 to     

Sinomenine influences capacity for invasion and migration in activated human monocytic THP-1 cells by inhibiting the expression of MMP-2, MMP-9, and CD147

Aim： The aim of this study was to investigate the mechanism of the effects of Sinomenine （SIN） on the invasion and migration ability of activated human monocytic THP-1 cells （A-THP-1）. Sinomenine is a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum.
Methods： Human monocytic THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate （PMA）. Cells were treated with different concentrations of SIN. The invasion and migration ability of cells was tested by in vitro transwell assays. The levels of CD147 and MMPs were evaluated by flow cytometric analysis and zymographic analysis, respectively. The mRNA expression of CD147, MMP-2, and MMP-9 was measured by RT-PCR.
Results： The invasion and migration ability of A-THP-1 cells was significantly inhibited by SIN in a concentration-depen- dent fashion; at the same time, the levels of CD147, MMP-2, and MMP-9 were markedly down-regulated. This inhibitory effect was most notable at concentrations of 0.25 mmol/L and 1.00 mmol/L （P〈0.01）.
Conclusion： A possible mechanism of the inhibitory effect of SIN on cell invasion and migration ability is repression of the expression of MMP-2 and MMP-9, which strongly correlates with the inhibition of CD147 activity.     

Role and possible mechanism of estrogen receptor α down-regulation leading to damage of TM4 Sertoli cell connectivity in mice北大核心CSCD

Aim To investigate the role of autophagy in the dysfunction of testicular TM4 cell junction induced by ERα down-regulation. Methods TM4 cells were treated with different concentrations of E R a inhibitor ICI182780 (ICI), and the proliferative activity of TM4 cells was detected by CCK-8 method. The number and morphological changes of TM4 cells were observed by light microscope. The levels of E R a, junction function related proteins and autophagy marker proteins were detected by Western blot. The expression and localization of Cx43 were detected by immunofluorescence staining. The cells were treated with chloroquine (CQ) and ICI for 24 h. The expression levels of autophagy and junction function related proteins were detected by Western blot. Results When ICI concentration was 50 nmol • L ~ or above, the cell viability decreased significantly. The increase of cell vacuoles in ICI group was observed by light microscope. Compared with normal control group, the protein expression levels of E R a, ZO-1, occludin, claudin-11, p-catenin and Cx43 in ICI groups significantly dropped, while the expression levels of N-cadherin and E-cadherin had no significant changes; LC3 II significantly rose, while p62 expression significantly fell. The results of immunofluorescence showed that the fluorescence expression of Cx43 in ICI group decreased significantly, but the position of CX43 did not change significantly. Compared with ICI group, the expression levels of LC3 II, p62, Cx43, ZO-1 and β-Catenin significantly increased. Conclusions The down-regulation of E R a leads to damage of TM4 cell junction function, which may be related to the activation of autophagy. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

The effects and mechanisms of cytoplasmic Macrophage colony-stimulating factor (M-CSF) on the proliferation, migration and invasion of HeLa cells

Objective To explore the effects and mechanisms of cytoplasmic M-CSF on the proliferation,migration and invasion of HeLa cells.Methods Both pCMV/cyto/myc vector and pCMV/cyto/myc-M-CSF vector was transfected into HeLa-cell by transfectaimine.After screening by G418,the positive clones were amplified and confirmed by RT-PCR,Western blot and immunocytochemistry.The effect of cytoplasmic M-CSF on the proliferation of HeLa cells were analyzed by cell conuting and antisense oligonucleotides.The migration and invasion of cell was measured by in vitro Transwell assay and Matrigel-coated polycarbonate filters.The expression of cyclinE,cyclinD1/2/3,CDK2/4/6,Rac1,and matrix metalloproteinase 2 and 9(MMP2/9)were assayed by semiquantitative RT-PCR.And expression of both α-tubulin and cdc42 were displayed by immunofluorescence.The activity of MMP2 was detected by gelatin zymography.Results Results A cell line(referred as to HeLa-M cell)that highly expresses cytoplasmic M-CSF was successfully established in the test.Our result indicated that HeLa-M cell had a larger volume,faster growth rate and shorter doubling time than either pCMV/cyto/myc transfected HeLa cells(referred as to HeLa-C cell)or untransfected HeLa cells(referred as to HeLa cell).M-CSF-specific antisense oligonucleoside significantly inhibited HeLa-M cell proliferation and had little effect on either HeLa-C cell or HeLa-C cell growth.Cytoplasmic M-CSF up-regulated both the expression of cyclinE,cyclinD1 and cyclinD3,CDK2,CDK 4 and CDK6,a Rho GTPase ralative protein(Rac1),cdc42 and MMP2,but had little effect on expression of MMP9 and cyclin D2.Furthermore,cytoplasmic M-CSF induced the rearrangement of the α-tubulin in HeLa cells and significantly promoted the migration and invasion of HeLa cells in vitro.Conclusions Cytoplasmic M-CSFs up-regulate the expression of cyclinE,cyclinD1 and cyclinD3,CDK2,CDK 4 and CDK6 and induces the proliferation of HeLa cells.Cytoplasmic M-CSFs up-regulate the expression of Rac1 and cdc42 and cause the rearrangement of the α-tubulin in HeLa cells.Furthermore Cytoplasmic M-CSFs increase both the expression and activity of MMP2 and promote the migration and invasion of HeLa cell in vitro.But cytoplasmic M-CSFs have little effect on expression of cyclin D2 and MMP9.     

Effect of curcumin on proliferation and cycle of human ECa-109 cells

OBJECTIVE To investigate the mechanism of curcumin on Inhibition of cell proliferation and apoptosis induction in esophageal cancer cells.METHODS Eca-109 cell growth inhibiting rates induced by curcumin were investigated by using CCK-8 assay.Eca-109 cell growth inhibiting rates induced by curcumin were investigated by using DNA gel electrophoresis for detection of DNA Ladder,electron microscopy observation technology for detection of ultrastructure and automatic fluorescence microplate reader.RESULTS Curcumin can inhibit cell proliferation growth inhibition rate had increased in a dose-and time-dependent manner.Caspase 3 activity assay detected that OD value with 20 μmol·L-1,40 μmol·L-1 of curcumin for 24 h,compared with the control group,the difference was statistically significant(P<0.05).Electron microscopy showed that curcumin can damaging cellular ultrastructure.CONCLUSION Curcumin can damage cellular ultrastructure of Eca-109 cells,Inhibit cell proliferation and induce its apoptosis.     

Yhhu3813 is a novel selective inhibitor of c-Met kinase that inhibits c-Met-dependent neoplastic phenotypes of human cancer cells

Aim： c-Met kinase deregulation is strongly associated with the formation, progression and dissemination of human cancers. In this study we identified Yhhu3813 as a small-molecule inhibitor of c-Met kinase and characterized its antitumor properties both in vitro and in vivo.
Methods： The activities of different kinases were measured using ELISA assays and signaling proteins in the cells were detected with Western blotting. Cell proliferation was assessed using SRB or MTT assay in twenty human cell lines and cell cycle distribution was determined with flow cytometry. Transwell-based assay was used to evaluate cell migration and invasion. Cell invasive growth was detected by a morphogenesis assay. c-Met overactivated human NSCLC cell line EBC-1 xenografts were used to evaluate the in vivo anti-tumor efficacy.

Results： Yhhu3813 potently inhibited c-Met kinase activity in vitro with an IC50 value of 2.4±0.3 nmol/L, 〉400-fold higher than that for a panel of 15 different tyrosine kinases, suggesting a high selectivity of Yhhu3813. The compound （20, 100 and 500 nmol/L） dose-dependently inhibited the phosphorylation of c-Met and its key downstream Akt and Erk signal cascades in multiple c-Met aberrant human cancer cell lines, regardless of the mechanistic complexity in c-Met activation across different cellular contexts. In 20 human cancer cell lines harboring different backgrounds of c-Met expression/activation, Yhhu3813 potently inhibited c-Met-driven cell proliferation via arresting cells at G1/S phase. Furthermore, Yhhu3813 substantially impaired c-Met-mediated cell migration, invasion, scattering, and invasive growth. Oral administration of EBC-1 xenograft mice with Yhhu3813 （50 or 100 mg·kg-1·d-1, qd, for 2 weeks） dose-dependently suppressed the tumor growth, which was correlated with a reduction in the intratumoral proliferation index and c-Met signaling.

Conclusion： Yhhu3813 is a potent selective inhibitor of c-Met that inhibits c-Met-dependent neoplastic phenotypes of human cancer cells in vitro and in vivo.     

Oxidative modulation of marcaine and lekoptin in H9C2 rat myoblasts

Aim： The cytotoxicity of marcaine was estimated in combination with a calcium channel blocker. In addition, the influence of marcaine and marcaine plus lekoptin on a model system using the H9C2 cardiac cell line was investigated. Methods： Cells were incubated for five hours with marcaine, lekoptin, or with both drugs simultaneously. Apoptotic cells were detected using the TUNEL assay and the alkaline comet assay. Mitochondrial cell function after drug uptake was examined using the MTT assay. The concentration of MDA （malondialdehyde） - the final product of fatty-acid peroxidation, was quantified spectrophotometrically. The expression of glutathione S-transferase n （GST-rI） was detected by immunofluorescence （IF） and Western blotting （WB） and inducible nitric oxide synthase （iNOS） was assessed by immuno-cytochemical staining （ABC）. Results： Incubation with marcaine resulted in the highest number of apoptotic cells. After incubation with both marcaine and lekoptin, moderate damage to cells （54.2%＋1.775% of DNA destruction） was observed. The highest levels of iNOS and GST-n expression were observed in cells treated with marcaine and marcaine plus lekoptin. The characteristic nuclear GST-n expression was observed in cells treated with both drugs. Conclusion： Lekoptin stimulated cells to proliferate. Marcaine caused membrane damage and ultimately cell death.     

Targeting fibroblast activation protein inhibits endothelial-mesenchymal transition by affecting cancer-associated fibroblasts derived exosomes北大核心CSCD

Aim To investigate whether targeted inhibition of fibroblast activation protein (FAP) can inhibit the endothelial-to-mesenchymal transition (EndMT) of vascular endothelial cells by affecting exosomes (Exo) of cancer-associated fibroblasts (CAFs) and explore the underlying mechanisms. Methods Primary CAFs and peri-tumor fibroblasts (PTFs) were obtained from lung cancer and peri-cancer tissues, and CAFs-exo and PTFs-exo were collected from culture medium, respectively. Exosomes from CAFs treated with specific FAP inhibitor (3.3 nmol • L-1 SP13786) for 24 h were named as Anti-FAP-exo. HMEC-1 cells were incubated in equal volumes of RPMI 1640, PTFs-exo, CAFs-exo and anti-FAP-exo respectively and named as control group, PTF group, C AF group and anti-FAP group. The scratch assay, Transwell invasion assay and angiogenesis assay were used to detect the migration ability, invasion ability and angiogenesis ability of HMEC-1 cells. Immunofluorescence, immunohistochemistry and Western blot were used to detect EndMT-associated protein expression. Results The migration ability, invasion ability and angiogenesis ability of HMEC-1 cells of CAF group were significantly higher than those of PTF group, whereas there was no significant difference between that of anti-FAP group and PTF group. HMEC-1 cells of CAF group had higher expression of α-SMA, SM22α, p-Stat3 and Snail, and lower expression of CD31 and VE-cadherin than that of PTF group. In addition, HMEC-1 cells of Anti-FAP group had lower expression of α-SMA, SM22α, pStat3 and Snail, and higher expression of CD31 and VE-cadherin than that of CAF group. Conclusions Specific inhibition of FAP could indirectly inhibit the migration ability, invasion ability and angiogenesis ability of vascular endothelial cells via affecting CAFs-exo and Stat3-snail-EndMT pathway may be the potential mechanism. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Inhibitory effects of lithospermic acid on proliferation and migration of rat vascular smooth muscle cells

Aim： To understand the effects of lithospermic acid （LA）, a potent antioxidant from the water-soluble extract of Salvia miltiorrhiza, on the migration and proliferation of rat thoracic aorta vascular smooth muscle cells （VSMCs）. Methods： VSMC migration, proliferation, DNA synthesis and cell cycle progression were investigated by transwell migration analysis, 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, bromodeoxyuridine （BrdU） incorporation assay, and flow cytometric detection, respectively. Intracellular reactive oxygen species （ROS） generation was detected using 2＇,7＇-dichlorofluorescin diacetate （DCFH-DA）. The expression of cyclin D1 protein and matrix metalloproteinase-9 （MMP-9） protein, as well as the phosphorylation state of ERK1/2, were determined using Western blots. The activity of MMP-9 and the expression of MMP-9 mRNA were assessed by gelatin zymography analysis and RT-PCR, respectively. Results： LA （25-100 pmol/L） inhibited both lipopolysaccharide （LPS）- and fetal bovine serum （FBS）-induced ROS generation and ERK1/2 phosphorylation. By down-regulating the expression of cyclin D1 and arresting cell cycle progression at the G1 phase, LA inhibited both VSMC proliferation and DNA synthesis as induced by 5% FBS. Furthermore, LA attenuated LPS-induced VSMC migration by inhibiting MMP-9 expression and its enzymatic activity. Conclusion： LA is able to inhibit FBS-induced VSMC proliferation and LPS-induced VSMC migration, which suggests that LA may have therapeutic effects in the prevention of atherosclerosis, restenosis and neointimal hyperplasia.     

Peplomycin induces G1-phase specific apoptosis in liver carcinoma cell line Bel-7402 involving G2-phase arrest

AIM: To investigate the mechanism of peplomycin (PEP)-induced apoptosis in liver carcinoma cell line (Bel-7402).METHODS: Growth inhibition by PEP was analyzed using 3- 4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazoliumbromide (MTT) assay. Apoptotic cells were detected using Hoechest 33258 staining, and confirmed by flowcytometric analysis and DNA fragmentation analysis. The expression of cyclin A and B 1 were determined by flowcytometry and Western blot. Annexin V assay was measured by flow cytometric analysis. RESULTS: PEPinduced apoptosis and then inhibited cell proliferation in liver carcinoma cell line Bel-7402. Cells treated with PEP50 μmol/L for 15 h were arrested in G2-phase with dramatical expression of cyclin A and a little change in cyclin B 1.Almost all the apoptosis occurred in cells undergoing the G1-phase after treatment for 24 h. CONCLUSION:Peplomycin induced G1-phase specific apoptosis in Bel-7402 involving G2-phase arrest.     

FKBP38 regulates apoptosis of dopaminergic neurons北大核心CSCD

Aim To investigate the role of FKBP38 in inhibiting apoptosis in a rotenone-induced Parkinson's disease(PD)cell model. Methods In vivo experiments:MPTP-induced PD in vivo models were constructed,and the expressions of α-synuclein,TH and FKBP38 in brains of PD mice were detected. In vitro experiments:Dopaminergic neuron MN9D cells were stimulated with rotenone to construct an in vitro model of PD; Western blot was used to detect the expression levels of α-synuclein,TH,Tom20 and FKBP38 in PD in vitro model; FKBP38 lentivirus was transferred into MN9D cells to construct stable overexpression and FKBP38 knockdown cell lines; CCK-8 assay was used to detect the cell viability of FKBP38 overexpression and knockdown cells stimulated by rotenone; Western blot was used to detect anti-apoptotic protein Bcl-2 and apoptosis protein in PD cell model expression levels of Bax. Results The expression level of FKBP38 was significantly down-regulated in both in vitro and in vivo models of PD(P<0.01). Knockdown of FKBP38 aggravated the decline of dopaminergic neuron cell viability caused by rotenone(P<0.05),while overexpression of FKBP38 significantly ameliorated the decline of dopaminergic neuron cell viability caused by rotenone(P<0.05). Western blot results showed that overexpression of FKBP38 could significantly up-regulate the expression level of anti-apoptotic protein Bcl-2 and increase the ratio of Bcl-2/Bax in PD dopaminergic neurons(P<0.05). Conclusion In the PD cell model regulation of FKBP38 can improve the apoptosis of dopaminergic neurons. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Astilbic acid induced COLO 205 cell apoptosis by regulating Bcl-2 and Bax expression and activating caspase-3

AIM: To investigate the effect of astilbic acid (3β, 6β-dihydroxyolean-12-en-27-oic acid, AA) on human colorectal carcinoma COLO 205 cell proliferation and apoptosis. METHODS: Proliferation of COLO 205 cells was measued by MTT assay. Content of DNA in COLO 205 cell was measued by modified diphenylamine assay. AA-induced morphological changes was observed with fluorescence microscope and transmission electron microscope. DNA fragmentation was visualized by agarose gel electrophoresis. Apoptosis rate and cell cycle distribution were determined by flow cytometric analysis. Expressions of Bcl-2 and Bax proteins were visioned by immunohistochemical analysis. The change of relative mitochondral transmembrane potential (MTP) in COLO 205 cell was analyzed with FCM after rhodamine 123 staining. RESULTS: The IC50 (96 h) of AA for inhibiting COLO 205 cell proliferation was 61.56 0.34 μmol/L. AA induced a marked concentration- and time-dependent inhibition of COLO 205 cell proliferation and reduced the DNA content in COLO 205 cell. Cells treated with AA 64 μmol/L showed typical morphological changes of apoptosis and DNA “ladder“ pattern. The cell cycle was arrested in G0/G1 phase, and the apoptosis rate was 28.25 % for COLO 205 cells treated with AA 64 μmol/L for 48 h. Meanwhile the expression of Bcl-2 protein was decreased while that of Bax was increased and relative MTP was decreased as well. DEVD-CHO1 μmol/L could increase the viability of COLO 205 cells treated with AA for 48 h. CONCLUSION: AA showed potent inhibitory activity on COLO 205 cells proliferation, and could induce COLO 205 cells apoptosis through disturbing DNA replication, down-regulating Bcl-2 expression, and up-regulating Bax expression, lowering relative MTP, and activating caspase-3 pathway.     

Dihydroartemisinin induces apoptosis and down- regulates hypoxia inducible factor- 1 α in C6 glioma cells

Astrocytoma is the most malignant tumor in brain without effective therapeutic drugs. Recently, artemisinin and its derivative dihydroartemisinin （DHA） were found to be active against the growth of some cancer cell lines. In the present study, we determined whether DHA exerts cytotoxic effect on C6 glioma cells, if so, what are the possible mechanisms. We found that DHA concentration - and time - dependently inhibited the growth of C6 cells as detected by trypan blue dye exclusion assay and cell proliferation assay; the IC50 value was 7.34 μM after 72 - h exposure. The cytotoxicity of DHA was enhanced by ferrous ions. DAH also induced the apoptosis and increased intracellular reactive oxygen species at 5 - 25 μM. This effect was associated with the increase of oxygen species by DHA. Immunocytochemistry and immunoblotting analysis revealed a significant reduction in the expression of hypoxia inducible factor - 1α under hypoxia conditions. These results indicate that DHA exerts an inhibitory effect on C6 cells by increasing the oxygen species and down - regulating expression of hypoxia inducible factor - 1α. Thus, DHA may be a potential therapeutic agent for the treatment of glioma.