Effects of diosgenin on autophagy of human osteosarcoma cells / 中成药

AIM To investigate the effects of diosgenin on autophagy of human osteosarcoma cells.METHODS Human osteosarcoma MG63 and U2OS cells with or without exposure to diosgenin had their proliferation detected by MTT assay,their ultrastructure observed by transmission electron microscopy,their expression of autophagy protein Beclin1 observed by immunofluorescence staining,and their expressions of autophagy molecular markers LC3,Beclin1 and PI3K/Akt/mTOR signaling pathway related proteins detected by Western blot.The MG63 and U2OS cells cotreated with diosgenin and PI3K pathway inhibitor LY294002 had the expression of Beclin1 mRNA detected by RT-qPCR.The MG63 and U2OS cells cotreated with autophagy inhibitor 3-methyladenine(3-MA)had their inhibition rate of proliferation detected by MTT assay,their expression of cleaved-caspase3 protein detected by Western blot,and their expression of caspase3 mRNA detected by RT-qPCR.RESULTS Upon osteosarcoma MG63 and U2OS cells,diosgenin inhibited their proliferation,promoted the generation of autophagosomes,increased the protein expression of LC3 Ⅱ and Beclin1(P<0.05,P<0.01),reduced the protein expression of LC3 I(P<0.01),and inhibited the protein phosphorylation level of PI3K/Akt/mTOR pathway(P<0.05,P<0.01),whose effects were offset by the intervention with autophagy inhibitors in terms of the reduced proliferation inhibition and down-regulated expressions of caspase3 mRNA and cleaved-caspase3 protein(P<0.01).CONCLUSION Diosgenin can inhibit the proliferation of osteosarcoma cells and induce their autophagy leading to their death and autophagy apoptosis,which may be related to the activation of PI3K/Akt/mTOR signaling pathway and up-regulation of the expression of LC3 Ⅱ and Beclin1 proteins.

miR-125b-5p inhibits proliferation and migration of osteosarcoma cells by negatively regulating RAB3D expression / 南方医科大学学报

OBJECTIVE@#To investigate the inhibitory effect of miR-125b-5p on proliferation and migration of osteosarcoma and the role of RAB3D in mediating this effect.@*METHODS@#The expression level of miR-125b-5p was detected by qRT-PCR in a normal bone cell line (hFOB1.19) and in two osteosarcoma OS cell lines (MG63 and HOS). A miR-125b-5p mimic or inhibitor was transfected in the osteosarcoma cell lines via liposome and the changes in cell proliferation and migration were detected with EDU and Transwell experiments. Bioinformatic analysis was conducted for predicting the target gene of miR-125b-5p, and the expression level of RAB3D in hFOB1.19, MG63, and HOS cells was detected by Western blotting. In the two osteosarcoma cell lines transfected with miR-125b-5p mimic or inhibitor, the expression levels of RAB3D mRNA and protein in osteosarcoma cells were examined with qRT-PCR and Western blotting. The effects of RAB3D overexpression, RAB3D knockdown, or overexpression of both miR-125b-5p and RAB3D on the proliferation and migration of cells were assessed using EDU and Transwell experiments.@*RESULTS@#The two osteosarcoma cell lines had significantly lower expression levels of miR-125b-5p (P < 0.05). Bioinformatic analysis predicted that RAB3D was a possible target gene regulated by miR-125b-5p. In osteosarcoma cells, overexpression of miR-125b-5p significantly lowered the expression of RAB3D protein (P < 0.05); inhibiting miR-125b-5p expression significantly decreased RAB3D expression only at the protein level (P < 0.05) without obviously affecting its mRNA level. Modulation of miR-125b-5p and RAB3D levels produced opposite effects on proliferation and migration of osteosarcoma cells, and in cells with overexpression of both miR-125b-5p and RAB3D, the effect of RAB3D on cell proliferation and migration was blocked by miR-125b-5p overexpression (P < 0.05).@*CONCLUSION@#Overexpression of miR-125b-5p inhibits the proliferation and migration of osteosarcoma cells by regulating the expression of RAB3D at the post-transcriptional level.

The role of autophagy and mitogen-activated protein kinase in cisplatin chemoresistance of osteosarcoma cells / 中国医师进修杂志

Objective@#To analyze the roles of autophagy and mitogen-activated protein kinase(MAPK) in cisplatin chemotherapy resistance of osteosarcoma cells.@*Methods@#The appropriate concentration of cisplatin was determined by clonogenic assay and the cisplatin-resistant osteosarcoma cells were gained. Western Blot was used to detect changes in the expression of autophagy and MAPK signaling. RT-PCR was used to detect changes in autophagy related gene transcription levels, and AnnexinV-FITC and Z-VAD-FMK was used to detect apoptosis. The contribution of drug inhibition of autophagy and MAPK signaling pathway in drug resistance of osteosarcoma cells was explored.@*Results@#Cell cloning assay showed that 7.5 μmol/L cisplatin concentration induced significant apoptosis. Drug-resistant cell lines were obtained through continuous drug screening of 10 d. Morphological changes of osteosarcoma cells after drug resistance were observed under microscope. At the same time, the key factor of DNA repair PARP protein expression was upregulated significantly. RT-PCR and Western Blot results showed that cisplatin activated autophagy in osteosarcoma cells. The levels of ATG5 and LC3B-Ⅱ protein and mRNA were upregulated and significantly different (P<0.05). Meanwhile, AnnexinV-FITC and Z-VAD-FMK results showed that apoptosis was significantly increased after inhibition of autophagy (P<0.05). Osteosarcoma cells restored the sensitivity to cisplatin.And this process had the participation of MAPK signal.@*Conclusions@#Autophagy and MAPK signal play an important role at cisplatin resistance in osteosarcoma cells, and the results can provide strategies for improving the treatment with the osteosarcoma patients.

JPH203, a selective L-type amino acid transporter 1 inhibitor, induces mitochondria-dependent apoptosis in Saos2 human osteosarcoma cells

Most normal cells express L-type amino acid transporter 2 (LAT2). However, L-type amino acid transporter 1 (LAT1) is highly expressed in many tumor cells and presumed to support their increased growth and proliferation. This study examined the effects of JPH203, a selective LAT1 inhibitor, on cell growth and its mechanism for cell death in Saos2 human osteosarcoma cells. FOB human osteoblastic cells and Saos2 cells expressed LAT1 and LAT2 together with their associating protein 4F2 heavy chain, but the expression of LAT2 in the Saos2 cells was especially weak. JPH203 and BCH, a non-selective L-type amino acid transporter inhibitor, potently inhibited L-leucine uptake in Saos2 cells. As expected, the intrinsic ability of JPH203 to inhibit L-leucine uptake was far more efficient than that of BCH in Saos2 cells. Likewise, JPH203 and BCH inhibited Saos2 cell growth with JPH203 being superior to BCH in this regard. Furthermore, JPH203 increased apoptosis rates and formed DNA ladder in Saos2 cells. Moreover, JPH203 activated the mitochondria-dependent apoptotic signaling pathway by upregulating pro-apoptotic factors, such as Bad, Bax, and Bak, and the active form of caspase-9, and downregulating anti-apoptotic factors, such as Bcl-2 and Bcl-xL. These results suggest that the inhibition of LAT1 activity via JPH203, which may act as a potential novel anti-cancer agent, leads to apoptosis mediated by the mitochondria-dependent intrinsic apoptotic signaling pathway by inducing the intracellular depletion of neutral amino acids essential for cell growth in Saos2 human osteosarcoma cells.

Study on osteosarcoma targeting of solid lipid nanoparticles modified with peptides specifically binding to human osteosarcoma cells / 中国生化药物杂志

Objective To prepare SLT peptide modified solid lipid nanoparticles(SLT-SLNs),study on its affinity to MG63 cell and tumor targeting.Methods Cellular uptake test was used to evaluate the uptake efficiency of MG63 cell for SLT-SLNs.MG63 cell were xenografted into athymic nude mice to establish the animal model,vivo imaging was used to evaluate the targeting efficiency. Results The average particle size of SLT-SLNs was (115.7 ±8.7)nm,polydispersity coefficient was 0.12,and Zeta potential was(13 ±2.25)mV.Cellular uptake test results showed that uptaken efficiency of SLT modified SLNs by MG63 cell were 4.4 times higher than that of SLNs.The fluorescence intensity of SLT-SLNs was much stronger than that of SLNs in vivo.Conclusion The SLT peptide modified solid lipid nanoparticles has a good osteosarcoma targeting efficiency,and it might serve as a promising osteosarcoma delivery system of antitumor drugs.

The effect of 50 Hz sinusoidal electromagnetic fields on the proliferation of the osteosarcoma cell line MG-63 / 中华物理医学与康复杂志

Objective To investigate the biological effect of 50 Hz sinusoidal electromagnetic fields at 1 mT on the proliferation of the human osteosarcoma cell line MG-63.Methods Osteosarcrma MG-63 cells were divided into control and experimental groups.The control group was incubated without an electromagnetic field; the experimental group was incubated in a 50 Hz,1.0 mT sinusoidal electromagnetic field.On the 2nd,4th and 6th day,their proliferation was determined using a cell counting kit-8(CCK-8)assay.Variations in the cell cycle were detected with flow cytometry(FCM).Semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR)was used to measure cyclin B1 and cyclin D1 mRNA.Results Compared with the control group,proliferation of the experimental group cells was reduced significantly.The percentage of cells at G0-G1 phase increased,and the mRNA expression of cyclin B1 and cyclin D1 was significantly reduced.Conclusions A 50 Hz sinusoidal electromagnetic field at 1.0 mT can inhibit the proliferation of osteosarcoma cell line MG-63 significantly.

Effect of recombinant human WN-α and cisplatin on cell apoptosis and invasion in MG-63 cell / 中华微生物学和免疫学杂志

of MG-63 cells to cisplatin,and can inhibit the invasion and metastasis of MG-63 cell.

Chios Gum Mastic Induces Cell Cycle Arrest and Apoptosis in Human Osteosarcoma Cells / 대한해부학회지

Chios gum mastic (CGM) is a resin produced from the stem and leaves of Pistiacia lentiscus L var chia, a plant which grows only on Chios Island in Greece. CGM has been used for many centuries as a dietary supplement and folk medicine for stomach and duodenal ulcers in many Mediterranean countries and is also known to induce cell cycle arrest and apoptosis in some cancer cells. This study was undertaken to investigate the alteration of the cell cycle and induction of apoptosis by CGM treatment on human osteosarcoma (HOS) cells. The viability and the growth inhibition of HOS cells were assessed by the MTT assay and clonogenic assay respectively. The hoechst staining, TUNEL assay and DNA electrophoresis were conducted to observe the HOS cells undergoing apoptosis. HOS cells were treated with CGM, and Western blotting, immunocytochemistry, confocal microscopy, FACScan flow cytometry, mitochondrial membrane potential change and proteasome activity were conducted. CGM treatment of HOS cells was found to result in a dose- and time-dependent decrease in cell viability, a dose-dependent inhibition of cell growth, and apoptotic cell death. Tested HOS cells also showed several lines of apoptotic manifestation and G1 arrest in cell cycle progression. In summary, this study clearly demonstrated that CGM induces G1 cell cycle arrest via the modulation of cell cycle-related proteins, and apoptosis via proteasome, mitochondrial and caspase cascades in HOS cells. Therefore, our data provide the possibility that a natural product, CGM could be considered as a novel therapeutic strategy for human osteosarcoma.

Apoptotic Effect of Co-Treatment with a Natural Product, Chios Gum Mastic, and a Synthetic Chenodeoxycholic Acid Derivative, HS-1200, on Human Osteosarcoma Cells / 체질인류학회지

Chios gum mastic (CGM) is a resinous exudate obtained from the stem and the main leaves of Pistacia lenticulus tree native to Mediterranean areas. Recently it reported that CGM induced apoptosis in a few cancer cells in vitro. It has been reported that the synthetic chenodeoxycholic acid (CDCA) derivatives showed apoptosis-inducing activity on various cancer cells in vitro. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with a natural product, CGM and a CDCA derivative, HS-1200 on human osteosarcoma (HOS) cells. To investigate whether the co-treatment of CGM and HS-1200 compared with each single treatment efficiently reduced the viability of HOS cells, MTT assay was conducted. Induction and augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining and DNA hypoploidy, Westen blot analysis and immunofluorescent staining were performed to study the alterations of the expression level and translocation of apoptosis-related proteins in co-treatment. Furthermore, proteasome activity and mitochondrial membrane potential (MMP) change were also assayed. In this study, HOS cells co-treated with CGM and HS-1200 showed several lines of apoptotic manifestation whereas each single treated HOS cells did not. Although the single treatment of 40 microgram/mL CGM or 25 micrometer HS-1200 for 24 h did not induce apoptosis, the cotreatment of them induced prominently apoptosis. Therefore our data provide the possibility that combination therapy of CGM and HS-1200 could be considered as a novel therapeutic strategy for human osteosarcoma.

Apoptotic Effect of Co-Treatment with a Natural Product, Chios Gum Mastic, and a Proteasome Inhibitor, Lactacystin, on Human Osteosarcoma Cells / 대한해부학회지

Chios gum mastic (CGM) is a resinous exudate obtained from the stem and the main leaves of Pistacia lenticulus tree native to Mediterranean areas. Recently, it was reported that CGM induced apoptosis in a few cancer cells in vitro. Since recent studies indicated the synergistic interactions between the apoptotic stimulus and a proteasome inhibitor, the ubiquintin-proteasome pathway has become an attractive target in cancer therapy. And to date, there has been no report of the synergistic apoptotic effect between CGM and a proteasome inhibitor to become an attractive target in cancer therapy. Therefore, this study was undertaken to investigate the synergistic apoptotic effect of co-treatment with a natural product, CGM, and a proteasome inhibitor, lactacystin, on human osteosarcoma (HOS) cells. To investigate whether the co-treatment of CGM and lactacystin compared with each single treatment efficiently induced apoptosis on HOS cells, MTT assay, DNA electrophoresis, Hoechst staining, DNA hypoploidy assay, Westen blot analysis, immunofluorescent staining, proteasome activity and mitochondrial membrane potential (MMP) change were performed. In this study, HOS cells co-treated with CGM and lactacystin showed several lines of apoptotic manifestation such as nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, the decrease of DNA content, the release of cytochrome c into cytosol, the translocation of AIF and DFF40 (CAD) onto nuclei, and activation of caspase-7, caspase-3, PARP and DFF45 (ICAD) whereas each single treated HOS cells hardly showed. We presented data indicating that the co-treatment of CGM and lactacystin induced potentially apoptosis whereas each single treatment did slightly. Moreover, the co-treatment of CGM and lactacystin potentiated the inhibition of proteasome activity. Therefore, our data provide the possibility that combination therapy of CGM and lactacystin could be considered as a novel therapeutic strategy for human osteosarcoma.

Screening for pathogenesis-related genes of osteosarcoma using gene microarray / 中国肿瘤生物治疗杂志

Objective:To screen for the pathogenesis-related genes of osteosarcoma and to assess their roles for the de- velopment of osteosareoma.Methods:Total RNA was extracted from 3 ATCC osteosarcoma cell lines and an osteoblastic cell line and was used to synthesize biotinylated cRNAs;the latter were hybridized to Affymetrix~(?)GeneChip~(?)U133A ar- rays and a gene with more than 2 folds of change was selected.Ten of the differentially expressed genes were chosen and the primers were designed and the synthesized.Then SYBR~(?)Green real-time PCR(RT-PCR)method was used to detect the expression of the 10 genes in 9 fresh osteosarcoma specimens.ABI Prism 7 000 system was used to analyze the differ- ent expression between osteosarcoma cell line and osteoblastic cell line.Results:We identified 58 up-regulated and 142 down-regulated genes in the 3 osteosareoma cell lines.Many of the genes were firstly reported to be related to the patho- genesis of osteosarcoma.These differentially expressed genes were mainly involved in energy and material metabolism,on- cogene,signal transduction gene,transcription- related genes,cell cycle-related genes,cell apoptosis-related gene,im- mune response gene,tumor suppressor genes,etc.The array results of 10 randomly selected genes were further verified by the RT-PCR in 9 fresh osteosarcoma specimens.Conclusion:Many genes are involved in the pathogenesis of osteosarcoma. Gene microarray can help to discover the genes related to the pathogenesis of osteosarcoma,which may lay a foundation for studying the molecular mechanism of osteosarcom.

Characteristics of Mitochondrial Events in Synthetic Bile Acids-induced Apoptosis of Human Osteosarcoma Cells / 체질인류학회지

ABSTRACT: Apoptosis of osteosarcoma cells induced by bile duct derivates, HS-1200 was investigated with relation to mitochodria. HS-1200 induced cytochrome c and Smac/DIABLO release from mitochondria which are major factors related to apoptosis. In these apoptosis processes, release of cytochrome c was not blocked by caspase inhibitor, but release of Smac/DIABLO was blocked. BKA, a kind of PTP (permeablity transition pore) inhibitor, did not block both of them. Interestingly, the alteration of MMP was not observed by means of using JC-1 dye. Although MitoTracker, DiOC-6 and Rhodamine123 were used to confirm previous results, the decrease of MMP was not observed. In order to investigate whether this phenomenon is apoptosis-specific or cell-specific process, genistein was added to cells which usually decreased MMP. After adding genistein, MMP was not decreased, suggesting this phenomenon is cell-specific process. Conclusionally, HS-1200 induced apoptosis of osteosarcoma cells via mitochondria, cytochrome c and Smac/DIABLO were released from mitochondria without decrease of MMP. The release of Smac/DIABLO was dependent of caspase.

Inhibition of curcumin on proliferation of human osteosarcoma U-2OS cells / 西安交通大学学报(医学版)

Objective To observe the inhibition of different concentrations of curcumin on cell growth,and explore the molecular mechanism of curcumin on human osteosarcoma.Methods Curcumin of different concentrations(0,10,20 and 40?mol/L) was administered to U-2OS cells of human osteosarcoma for 12h,24h and 48h,respectively.The proliferation of U-2OS cells was measured by MTT assay.Results Curcumin significantly inhibited the proliferation of PC-3 cells in vitro in concentration-and time-dependent manners.The higher the concentration of curcumin,the stronger the inhibition was(10?mol/L,20?mol/L,40?mol/L of r=-0.9638,-0.9778 and-0.9764,respectively,P

Synthetic Bile Acid Derivative HS-1200-induced Apoptosis of Human Osteosarcoma Cells / 대한해부학회지

Bile acids and synthetic its derivatives induced apoptosis in various kinds of cancer cells and had anticancer effects. However, it wasn`t discovered those materials have apoptosis induced effects on osteosarcoma cells. The present study was done to examine the synthetic bile acid derivatives induced apoptosis on osteosarcoma cells and such these apoptosis events. The synthetic bile acid derivatives, chenodeoxycholic acid (CDCA) induced the cell death on human osteosarcoma (HOS) cells contrary to ursodeoxycholic acid (UDCA). HS-1200, a synthetic derivative of CDCAs, was chosen to experiment apoptosis events in HOS cells. HOS cells treated with HS-1200 showed nucleus condensation, cytochrom c release, Bax/Bcl-xL alteration, activation of caspase-3 and caspase-activated deoxyribonuclease (CAD), and degradation of poly (ADP-ribose) polymerase (PARP). Though this study needs more investigations, these in vitro data suggest that treatment of the synthetic bile acid derivatives can give medical therapy on HOS cells.

Studies on anticancer effects of extracts caesalpinia sappan on oral carcinoma and osteosarcoma cells

Anticancer effect of methanol extract of Caesalpinia sappan L. on oral carcinoma (KB) and osteosarcoma (HOS) cells were investigated in this study. In order to elucidate the anticancer mechanism of Caesalpinia sappan L, we analyzed telomerase inhibitory effect of the methanol extract of Caesalpinia sappan L. In addition we prepared 5 fraction samples according to its polarity differences and analyzed anticancer effects on oral carcinoma and osteosarcoma cells. Following results are obtained in this study. 1. 50% cell proliferation inhibitory value (IC50) of the methanol extract of Caesalpinia sappan L. against oral carcinoma (KB) cells and osteosarcoma (HOS) cells were 9.0 microgram/ml and 10.9 microgram/ml, respectively. 2. The methanol extract of Caesalpinia sappan L. showed inhibitory effect of telomerase which is required for cancer cell immortality. Therefore, it seems that the anticancer effect of methanol extract of Caesalpinia sappan is at least partially due to telomerase inhibitory effect. 3. Five fraction samples were prepared according to its polarity and 88.7% of ingredient of total methanol extract was transferred to ethylacetate fraction. Thin layer chromatography analysis showed that dichloromethane fraction contained ingredient with relatively high polarity and ethylacetate fraction contained similar ingredient found in total methanol extract. 4. Anticancer effect was observed in n-hexane, dichloromethane, and ethylacetate fractions. The highest anticancer effect was found in dichloromethane fraction which had IC50 value of 4.4 and>4.0 microgram/ml against oral carcinoma (KB) cells and osteosarcoma (HOS) cells, respectively.

The Apoptosis Induction Effect of Recombinant Caspase-3 on the Human Osteosarcoma Cell Line SOSP-9901 / 中国医师杂志

Objective To investigate the apoptosis induction effect of recombinant caspase-3 expression on osteosarcoma cell line SOSP-9901. Methods Recombinant caspase-3 gene was subcloned into the GFP reporter vector pEGFP-C1 to generate the expression vector pEGFP-caspase-3 by DNA recombinant technique. pEGFP-caspase-3 was transfected into human osteosarcoma cell line SOSP-9901 by Lipofectamine 2000. The expression of recombinant caspase-3 was determined by RT-PCR. The morphological changes of transfected cells were observed under flurescent and electronic microscope. The cell survival rate of transfected cells was assayed by MTT method. Results Recombinant caspase-3 gene could be stably expressed in the transfected SSOP-9901 cells. Recombinant caspase-3 could obviously induce SOSP-9901 apoptosis, and inhibit SSOP-9901 cell proliferation in vitro. Conclusion Recombinant caspase-3 could inhibit the growth of the osteosarcoma cell line SOSP-9901 and induce it into apoptosis.

The Induction of hsp70 mRNA under Heat Stress / 第二军医大学学报

The induction of hsp70 mRNA in human osteosarcoma cells (HOS-8603) and intacl rats under heat stress was studied using hsp70 cDNA labeled with a-32P dATP. The results showed that the induction of hsp70 mRNA was evident in liver, lung, spleen and the most evident was found in brain when the core body temperature of rats was brought to 42℃ for 15 min. The induction of hsp70 mRNA in HOS-8603 was also significant when cells were cultured at 42℃ for 30 min. These results indicated that hsp70 mRNA could be induced both in vitro and in vivo under heat stress condition and the induction of hsp70 mRNA in intact rats was of tissue-specific fashion.