BMP6 reverses TGF-β1-induced changes in HK-2 cells： implications for the treatment of renal fibrosis

Aim： The aim of the study was to investigate the potential role of BMP6 in TGF-β1-mediated changes in HK-2 cells.
Methods： BMP6 was purified via heparin affinity and reverse phase liquid chromatography. The purity, specificity, and bioactivity of BMP6 were determined by SDS-PAGE, Western blot assays, and the induction of alkaline phosphatase （ALP） activity, respectively. Cell proliferation, morphology, and expression levels of α-SMA and E-cadherin were assessed by cell viability, microscopy, and Western blot assays, respectively. In addition, cell adhesion abilities were determined by counting the number of attached cells, The expression of fibroneetin, collagen Ⅳ, matrix metalloproteinases 2 （MMP-2）, and tissue inhibitors of matrix metalloproteinases 2 （TIMP-2） were analyzed using RT-PCR. MMP-2 activity was analyzed by zymography, whereas the activation of the MAPKs and Smad signaling were analyzed using Western blot assays and a reporter gene assay, respectively.
Results： Our results indicated that recombinant BMP6 induced ALP activity Jn a dose-dependent and time-course-dependent manner. Treatment with TGF-β1 reduced both the cell proliferation and the expression of E-cadherin, induced a morphological transformation, decreased the expression and activity of MMP-2, and increased the expression levelsof α-SMA, flbronectin, and TIMP-2 in HK-2 cells. All of these effects were inhibited when cells were treated with TGF-β1 in combination with rhBMP6, whereas rhBMP6 alone demonstrated no such effect. Treatment with TGF-β1, rhBMP6, or a combination of both had no effect on the expression of collagen IV. In addition, the administration of rhBMP6 prevented the enhanced adhesion behavior triggered by TGF-β1. Furthermore, the addition of rhBMP6 abrogated the JNK and Smad2/3 signaling that was activated by TGF-β1.
Conclusion： BMP6 ameliorated the TGF-131-induced changes in HK-2 cells. The suppression of TGF-β1-mediated JNK and Smad2/3 signaling activation were implicated in these effect     

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.     

Icaritin suppresses the proliferation of human osteosarcoma cells in vitro by increasing apoptosis and decreasing MMP expression

Aim： To explore whether icaritin, a prenylflavonoid derivative of the Chinese tonic herb Epimedium, could suppress the proliferation of human osteosarcoma cells in vitro, and to elucidate the mechanisms of the action.
Methods： Human osteosarcoma SaOS2 cell line was used in the present study. The proliferation of the cells was examined using MTT assay and immunofluorescence DAPI staining. Cell motility was studied with the scratch assay. Cell apoptosis was determined by Annexin V-FITC and PI double staining using flow cytometry. Western blotting and RT-PCR were used to measure the expression of mRNAs and proteins in the cells.

Results： Icaritin （5–15 μmol/L） suppressed the proliferation of SaOS2 cells in vitro in a dose-dependent manner. Furthermore, the cell motility was significantly decreased after exposure to icaritin. Moreover, icaritin （5 μmol/L） time-dependently induced the apoptosis of SaOS2 cells, markedly suppressed MMP-2 and MMP-9 expression, upregulated caspase-3 and caspase-9 expression, and increased the level of cleaved caspase-3 in the cells. Co-exposure to the caspase-3 inhibitor zVAD-fmk （10 μmol/L） compromised the icaritin-induced caspase-3 expression and apoptosis in SaOS2 cells.

Conclusion： Icaritin suppresses the proliferation of SaOS2 human osteosarcoma cells by increasing apoptosis and downregulating MMP expression.     

Adenovirus-mediated expression of UHRF1 reduces the radio- sensitivity of cervical cancer HeLa cells to γ-irradiation

Aim： An in vitro study was carried out to determine the effect of UHRF1 overexpression on radiosensitivity in human cervical cancer HeLa cells using adenovirus-mediated UHRF1 gene transfer （Ad5-UHRF1）.
Methods： Cell survival was evaluated using the clonogenic survival assay and the MTT assay; apoptosis and cell cycle distribution were monitored by flow cytometry. Protein levels were measured by Western blotting. Silencing XRCC4 expression was performed by transfection of small interfering RNA （siRNA）.
Results： Increased expression of UHRF1 by Ad5-UHRF1 significantly reduced the radiosensitivity of HeLa cells. The UHRF1-mediated radioresistance was correlated with increased DNA repair capability and increased expression of the DNA damage repair protein, XRCC4. Knocking down XRCC4 expression in the cells using XRCC4 siRNA markedly reduced the UHRF1-mediated radioresistance.
Conclusion： These results provide the first evidence for revealing a functional role of UHRF1 in human cervical cancer cells as a negative regulator of radiosensitivity.     

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 %     

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.     

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）.     

Oridonin induces NPM mutant protein translocation and apoptosis in NPM1c＋ acute myeloid leukemia cells in vitro

Aim： Skewed cytoplasmic accumulation of NPM mutant protein （NPM1c＋） is close related to leukemia pathogenesis. The aim of this study was to investigate whether oridonin, a diterpenoid isolated from the Chinese traditional medicine Rabdosia rubescens, was able to interfere with NPM1c＋ protein trafficking and induce apoptosis in NPM1c＋ acute myeloid leukemia cells in vitro.
Methods： OCI-AML3 cell line harboring a NPM1 gene mutation was examined. Cell growth was detected by MTT assay. Cell apoptosis was evaluated using flow cytometry and Hoechst 33258 staining. The expression and subcellular localization of relevant proteins were detected by Western blot and immunofluorescent staining. The mRNA expression was detected by RT-PCR.
Results： Oridonin （2–12 μmol/L） dose-dependently inhibited the viability of OCI-AML3 cells （the IC50 value was 3.27±0.23 μmol/L at 24 h）. Moreover, oridonin induced OCI-AML3 cell apoptosis accompanied by activation of caspase-3 and nuclear translocation of NPM1c＋ protein. Oridonin did not change the expression of Crm1 （the export receptor for nuclear export signal-containing proteins）, but induced nuclear translocation of Crm1. Oridonin markedly increased the expression of nucleoporin98 （Nup98）, which had an important role in Crm1-mediated nuclear protein export, and induced nuclear accumulation of Nup98. Furthermore, oridonin markedly increased the expression of p14arf and p53.
Conclusion： In NPM1c＋ leukemia cells, oridonin induces NPM1c＋ protein translocation into the nucleus possibly via nuclear accumulation of Crm1; the compound markedly increases p53 and p14arf expression, which may contribute to cell apoptosis.     

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.     

P53-mediated cell cycle arrest and apoptosis through a caspase-3- independent, but caspase-9-dependent pathway in oridonin-treated MCF-7 human breast cancer cells

Aim： To study the caspase-3-independent mechanisms in oridonin-induced MCF-7 human breast cancer cell apoptosis in vitro. Methods： The viability of oridonin- treated MCF-7 cells was measured by MTT （thiazole blue） assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. The apoptotic ratio was determined by lactate dehydrogenase assay. Cell cycle alternation and mitochondrial membrane potential were measured by flow cytometric analysis. Bax, Bcl-2, caspase-3, caspase-9, heat shock protein （Hsp）90, p53, p-p53, p21, Poly （ADP-ribose） polymerase （PARP）, and the inhibitor of caspase-activated DNase （ICAD） protein expressions were detected by Western blot analysis. Results： Oridonin inhibited cell growth in a time- and dose-dependent manner. Cell cycle was altered through the upregulation of p53 and p21 protein expressions. Pancaspase inhibitor Z-VAD-fmk and calpain inhibitor II both decreased cell death ratio. Nucleosomal DNA fragmentation and the downregulation of △ψmit were detected in oridonin-induced MCF-7 cell apoptosis, which was involved in a postmitochondrial caspase-9-dependent pathway. Decreased Bcl-2 and Hsp90 expression levels and increased Bax and p21 expression levels were positively correlated with elevated levels of phosphorylated p53 phosphorylation. Moreover, PARP was partially cleaved by calpain rather than by capase-3. Condusion： DNA damage provoked alternations in the mitochondrial and caspase-9 pathways as well as p53-mediated cell cycle arrest, but was not related to caspase-3 activity in oridonin-induced MCF-7 cells.     

Berbamine exhibits potent antitumor effects on imatinib-resistant CML cells in vitro and in vivo

Aim The aim of this study was to explore the effects and mechanism of berbamine on imatinib-resistant BCR-ABL-positive human leukemia K562 （K562-r） cells in vitro and in vivo. Methods：Cell viability was measured by MTT assay, and apoptotic morphology changes were detected by fluorescence microscopy. The apoptosis rate was measured by flow cytometric assay, mdr-1 mRNA levels were determined by RT-PCR. Bcl-2 family proteins, cytochrome c（ cyt C） , poly （ADP-ribose） polymerase （PARP）, and P-glycoprotein were detected by Western blot. BALB/c nu/nu mice were injected with K562-r cells subcutaneously. Tumor-bearing mice were treated intravenously with berbamine.
Results： MTT tests revealed that berbamine significantly inhibited K562-r cell proliferation and increased the chemosensitivity of K562-r cells to imatinib. The apoptosis rate was significantly increased following treatment with 21.2μmol/L berbamine; formation of typical apoptotic blebs was apparent, as observed by fluorescence microscopy. Expression levels of mdr-I mRNA and P-gp protein were high in untreated K562-r cells and significantly down-regulated by berbamine treatment. Berbamine-treated K562-r cells also exhibited down-regulated expression of the anti-apoptotic proteins Bcl-2 and Bcl-XL up-regulated expression of the apoptotic proteins Bax and cytoplasmic cyt C, and stimulated proteolytic cleavage of PARP. In addition, berbamine also suppressed the growth of K562-r xenotransplanted tumors in vivo.
Conclusion Berbamine inhibited proliferation of K562-r ceils both in vitro and in vivo. Berbamine-induced apoptosis in K562-r cells appeared to occur through a mechanism involving Bcl-2 family proteins, as well as mdr-1 mRNA and P-gp pro- tein. Berbamine in combination with imatinib restored the chemo-sensitivity of K562-r cells to imatinib. Our findings suggest that berbamine may be useful in treating imafinib-resistant CML patients.     

Ginsenoside Rgl promotes endothelial progenitor cell migration and proliferation

Aim： To investigate the effect of ginsenoside Rgl on the migration, adhesion, proliferation, and VEGF expression of endothelial progenitor cells （EPCs）.
Methods： EPCs were isolated from human peripheral blood and incubated with different concentrations of ginsenoside Rgl （0.1, 0.5, 1.0, and 5.0 μmol/L） and vehicle controls. EPC migration was detected with a modified Boyden chamber assay. EPC adhesion was determined by counting adherent cells on fibronectin-coated culture dishes. EPC proliferation was analyzed with the 3.（4,5.dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay. In vitro vasculogenesis was assayed using an in vitro vasculogenesis detection kit. A VEGF-ELISA kit was used to measure the amount of VEGF protein in the cell culture medium.
Results： Ginsenoside Rgl promoted EPC adhesion, proliferation, migration and in vitro vasculogenesis in a dose- and timedependent manner. Cell cycle analysis showed that 5.0 μmol/L ofginsenoside Rgl significantly increased the EPC proliferative phase （S phase） and decreased the resting phase （G0/G1 phase）. Ginsenoside Rgl increased vascular endothelial growth factor production. Conclusion： The results indicate that ginsenoside Rgl promotes proliferation, migration, adhesion and in vitro vasculogenesis.     

Inhibitory effect of 1,8-cineol （eucalyptol） on Egr-1 expression in lipopolysaccharide-stimulated THP-1 cells

Aim： To study the effects of 1,8-cineol （eucalyptol） on the expression of early growth response factor-1 （Egr-1） and NF-κB in the human monocyte THP-1 cell line stimulated by lipopolysaccharide （LPS）. Methods： The THP-1 cells were incu- bated with serial doses of 1,8-cineol （1, 10, and 100 mg/L, 30 min） before being stimulated with LPS （1 mg/L, 30 min）. The localization of Egr-1 in the THP-1 cells was detected by immunofluorescence and a laser scanning confocal microscope. The expression of Egr-1 in the nuclei and whole cell, and NF-κB in the nuclei, were measured by Western blot analysis. Results： When stimulated by LPS, the FITC- labeled Egr-1 was detected mainly in the nuclei. Moreover, the expression of Egr-1 in the whole cell increased markedly compared with the control cells. 1,8- Cineol pretreatment decreased the expression of Egr-1 in both the nuclei and whole cell of the LPS-stimulated THP- 1 cells, and this effect was concentration- dependent, but there was no reaction on the expression of NF-κB in the nuclei protein in the LPS-stimulated THP-1 cells. Conclusion： In a concentration-depen- dent manner, 1,8-Cineol reduces LPS-induced Egr-1 expression in nuclei and in whole cell of THP-1 cells, but shows no effect on NF-κB expression.     

Overexpression of coxsackie and adenovirus receptor inhibit growth of human bladder cancer cell in vitro and in vivo

Aim： To study the effect of the overexpression of coxsackie and the adenovirus receptor （CAR） on the growth of the human bladder cancer cell in vitro and in vivo. Methods： A retroviral vector pLXSN-CAR expressing CAR was constructed and confirmed by restriction enzyme mapping. The pLXSN-CAR vector and con- trol vector pLXSN were transfected into the PT67 packaging cell line to generate retrovirus with high titer. The CAR-negative T24 cell was infected with the pLXSNCAR and the pLXSN retrovirus, respectively. The positive clone cells were selected with G418 for 2 weeks. The expression level of the CAR protein was detected by Western blot assay. T24 cell growth in vitro was determined by 3-（4, 5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay. Anchorage-independent growth was measured by soft-agar colony formation assay. In vivo cell growth was determined by a nude mice xenograft model. Results： The pLXSN-CAR vector containing full-length CAR cDNA was successfully constructed. Western blot analysis showed that a 46 kDa specific band was found in pLXSN-CA-transfected T24 cells. MTT assay identified the growth inhibition of T24/pLXSN-CAR cells. The cell colony forming ability of T24/pLXSNCAR cells was significantly lower than that of T24/pLXSN and parental T24 cells. There was a reduction in the tumor size in the T24/pLXSN-CAR group as compared with that of the T24/pLXSN group and parental T24 group. Conclusion： The overexpression of CAR in T24 bladder cancer cells can inhibit cell growth both in vitro and in vivo.     

Trafficking of α1B-adrenergic receptor mediated by inverse agonist in living cells

AIM The project is aimed at understanding the action of inverse agonist at single molecule level and capturing the real time picture of molecular behavior of α1B-adrenergic receptor (AR) mediated by inverse agonist in living cells by single molecule detection (SMD). METHODS The location and distribution of α1B-AR was detected by laser confocal and whole cell ^3H-prazosin binding assay. Dynamic imaging of BODIPY-FL-labeled prazosin (Praz), specific antagonist of (1-AR, was observed in α1B-AR stably expressed human embryonic kidney 293 (HEK293) living cells. The detection of real-time dynamic behaviors of AR was achieved by using fluorescence-labeled AR and its ligand combined with SMD techniques. RESULTS α1B-AR was predominantly distributed on the cell surface and 8.2% of the total receptors were located in cytosol.