scholarly journals Down-regulation of XIAP enhances the radiosensitivity of esophageal cancer cells in vivo and in vitro

2017 ◽  
Vol 37 (5) ◽  
Author(s):  
Xin Wen ◽  
Xin-Rui Han ◽  
Shao-Hua Fan ◽  
Zi-Feng Zhang ◽  
Lu Chen ◽  
...  
Keyword(s):  
Esophageal Cancer ◽  
Gene Silencing ◽  
Cell Survival ◽  
Cell Lines ◽  
Western Blotting ◽  
Nude Mice ◽  
Caspase 3 ◽  
Control Group ◽  
Caspase 9 ◽  
Blank Control Group

The study investigated the effects of X-chromosome-linked inhibitor of apoptosis (XIAP) gene silencing on the radiosensitivity of esophageal cancer (EC) cells. Western blotting was used to select EC cell lines with XIAP overexpression. Selected EC9706 and KYSE30 cell lines were both divided into four groups: the blank control group, the negative control (NC) group (transfected with pBSHH1), the siRNA-enhanced group (transfected with pBSHH1-XIAP1-siRNA), and the siRNA-decreased group (transfected with pBSHH1-XIAP2-siRNA). Expressions of XIAP were measured by reverse-transcription quantitative PCR (RT-qPCR) and Western blotting, cell survival and viability by MTT assay and colony formation assay, and cell apoptosis by flow cytometry, respectively. Caspase-3 and caspase-9 activity were detected using caspase-3 and caspase-9 activity detection kits. A nude mice model of EC9706 cell line was established to measure tumorigenesis ability. Compared with the NC group, XIAP mRNA and protein expressions were decreased, caspase-3 and caspase-9 activity and apoptosis were up-regulated, and cell survival rate and colony-forming efficiency were lower in the siRNA-enhanced and siRNA-decreased groups in both the cell lines; while the opposite trends were found in the siRNA-decreased group compared with the siRNA-enhanced group. Tumor weight and volume of nude mice were decreased in the siRNA-enhanced and siRNA-decreased groups than those in the NC group, and were elevated in the siRNA-decreased group compared with the siRNA-enhanced group. These results indicate that XIAP gene silencing would strengthen the radiosensitivity of EC9706 cells, which provides a novel target for the treatment of EC.

scholarly journals Protein Arginine Methyltransferase 5 Promotes Esophageal Squamous Cell Carcinoma Proliferation and Metastasis via LKB1/AMPK/mTOR Signaling Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Yu-ru Chen ◽  
Hua-ni Li ◽  
Lian-jun Zhang ◽  
Chong Zhang ◽  
Jin-guang He
Keyword(s):  
Squamous Cell Carcinoma ◽  
Cell Migration ◽  
Esophageal Squamous Cell Carcinoma ◽  
Cell Lines ◽  
Western Blotting ◽  
Cell Apoptosis ◽  
Caspase 3 ◽  
Rt Pcr ◽  
Caspase 9 ◽  
Arginine Methyltransferase

Background: Esophageal squamous cell carcinoma (ESCC) is the eighth most common cancer in the world. Protein arginine methyltransferase 5 (PRMT5), an enzyme that catalyzes symmetric and asymmetric methylation on arginine residues of histone and non-histone proteins, is overexpressed in many cancers. However, whether or not PRMT5 participates in the regulation of ESCC remains largely unclear.Methods: PRMT5 mRNA and protein expression in ESCC tissues and cell lines were examined by RT-PCR, western blotting, and immunohistochemistry assays. Cell proliferation was examined by RT-PCR, western blotting, immunohistochemistry assays, MTT, and EdU assays. Cell apoptosis and cell cycle were examined by RT-PCR, western blotting, immunohistochemistry assays, and flow cytometry. Cell migration and invasion were examined by RT-PCR, western blotting, immunohistochemistry assays, and wound-healing and transwell assays. Tumor volume, tumors, and mouse weight were measured in different groups. Lung tissues with metastatic foci, the number of nodules, and lung/total weight were measured in different groups.Results: In the present study, the PRMT5 expression level was dramatically upregulated in ESCC clinical tissues as well as ESCC cell lines (ECA109 and KYSE150). Furthermore, knocking down PRMT5 obviously suppressed cell migration, invasion, proliferation, and cell arrest in G1 phase and promoted cell apoptosis in ESCC cells. Meanwhile, downregulating PRMT5 also increased the expression levels of Bax, caspase-3, and caspase-9, while expression levels of Bax-2, MMP-2, MMP-9, and p21 were decreased, which are members of the cyclin-dependent kinase family. Furthermore, knocking down PRMT5 could increase the expression of LKB1 and the phosphorylation (p)-AMPK expression and decrease the p-mTOR level. Additionally, overexpression of LKB1 could reveal anti-tumor effects in ESCC cell lines by inhibiting ESCC cell, migration, invasion, and proliferation and accelerating cell apoptosis. Besides, upregulating LKB1 expression could increase the levels of Bax, caspase-3, and caspase-9 and weaken the levels of Bax-2, MMP-2, and MMP-9. Moreover, knocking down PRMT5 could weaken the tumor growth and lung metastasis in vivo with upregulating the LKB1 expression and the p-AMPK level and downregulating the p-mTOR expression.Conclusion: PRMT5 may act as a tumor-inducing agent in ESCC by modulating LKB1/AMPK/mTOR pathway signaling.

scholarly journals Direct Effect of Septic Plasma in Human Cell Lines Viability

2018 ◽  
Vol 47 (1-3) ◽  
pp. 270-276
Author(s):  
Grazia Maria Virzì ◽  
Chiara Borga ◽  
Chiara Pasqualin ◽  
Silvia Pastori ◽  
Alessandra Brocca ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cell Viability ◽  
Cell Lines ◽  
Caspase 3 ◽  
Test Group ◽  
Organ Injury ◽  
Enzyme Linked Immunosorbent Assay ◽  
Caspase 9 ◽  
Renal Tubular Cells

Background: Sepsis is a life-threatening condition often associated with a high incidence of multiple organs injury. Several papers suggested the immune response by itself, with the production of humoral inflammatory mediators, is crucial in determining organ injury. However, little is known of how sepsis directly induces organ injury at the cellular levels. To assess this point, we set up an in vitro study to investigate the response of renal tubular cells (RTCs), monocytes (U937) and hepatocytes (HepG2) after 24 h-incubation with septic patients’ plasma. Methods: We enrolled 26 septic patients (“test” group). We evaluated cell viability, apoptosis and necrosis by flow cytometer. Caspase-3,-8,-9 and cytochrome-c concentrations have been analyzed using the Human enzyme-linked immunosorbent assay kit. Results: We found that a decrease of cell viability in all cell lines tested was associated to the increase of apoptosis in RTCs and U937 (p < 0.0001) and increase of necrosis in HepG2 (p < 0.5). The increase of apoptosis in RTCs and U937 cells was confirmed by higher levels of caspase-3 (p < 0.0001). We showed that apoptosis in both RTCs and U937 was triggered by the activation of the intrinsic pathway, as caspase-9 and cytochrome-c levels significantly increased (p < 0.0001), while caspase-8 did not change. This assumption was strengthened by the significant correlation of caspase-9 with both cytochrome-c (r = 0.73 for RTCs and r = 0.69 for U937) and caspase-3 (r = 0.69 for RTCs and r = 0.63 for U937). Conclusion: Humoral mediators in septic patients’ plasma induce apoptosis. This fact suggests that apoptosis inhibitors should be investigated as future strategy to reduce sepsis-induced organ damages.

Brucea javanica Oil Induces Apoptosis in T24 Bladder Cancer Cells via Upregulation of Caspase-3, Caspase-9, and Inhibition of NF-κB and COX-2 Expressions

2010 ◽  
Vol 38 (03) ◽  
pp. 613-624 ◽  
Author(s):  
Guo-Guang Lou ◽  
Hang-Ping Yao ◽  
Li-Ping Xie
Keyword(s):  
Caspase 3 ◽  
Flow Cytometric Analysis ◽  
Morphological Characteristics ◽  
Control Group ◽  
Caspase 9 ◽  
Brucea Javanica ◽  
Flow Cytometric ◽  
T24 Cells ◽  
Bladder Cancer Cells ◽  
Cox 2

The potential molecular mechanism of Brucea javanica oil in the induction of apoptosis of T24 bladder cancer cells was investigated in vitro. T24 cells were divided into two groups: one, treated with B. javanica oil and the other, untreated. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) and 4 mM glutamine. The morphological characteristics of T24 cells were examined microscopically at the 2nd and 5th day of the culture. The drug toxicity spectrum ( IC 50) was estimated by the MTT assay, and viability of T24 cells was assessed on the basis of the percentage of T24 apoptotic cells, as determined by Annexin/PI staining and flow cytometric analysis. The expression of caspase-3, capase-9, NF-κB p65, and COX-2 was analyzed by Western blotting. Morphological characteristics of the cells on the 2nd day showed apoptosis of the treated T24 cells; it was more apparent in the cells on the 5th day. B. javanica oil decreased the cell viability at the testing concentrations spectrum (5–0.156 mg/ml), and this viability was significantly higher as compared to the control group. In this concentration spectrum, B. javanica oil also induced apoptosis of T24 cells, which was analyzed by annexin/PI staining and flow cytometric analysis. These results were also statistically significant as compared to those of the control group. The expressions of caspase-3 and caspase-9 were low in the control T24 cells, while the expressions of NF-κB and COX-2 were high in normal T24 cells. Treatment with B. javanica oil significantly induced the expressions of caspase-3 and caspase-9 proteins in T24 cells, whereas the expressions of NF-κB and COX-2 proteins were inhibited. B. javanica oil significantly reduced the viability of T24 cells and induced T24 cell apoptosis. The molecular mechanism underlying these effects may be the activation of caspase apoptotic pathway by upregulation of the expression of caspase-3 and caspase-9 proteins and inhibition of the expression of NF-κB and COX-2 proteins.

scholarly journals Antiproliferative and Apoptotic Effects of Red Beetroot and Betanin on Human Colorectal Cancer Cell Lines

2021 ◽  
Author(s):  
Amir Saber ◽  
Nasim Abedimanesh ◽  
Mohammad-Hossein Somi ◽  
Ahmad Yari Khosroushahi
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Caspase 3 ◽  
Caspase 8 ◽  
Alcoholic Extract ◽  
Caspase 9 ◽  
Dapi Staining ◽  
Normal Cells ◽  
Colorectal Cancer Cell Lines ◽  
Ht 29

Abstract Background: Colorectal cancer (CRC) is the third most common type of cancer worldwide. Fruit and vegetables have some active compounds such as flavonoids and polyphenols that protect against malignancies through their antioxidative, anti-inflammatory, anti-proliferative, neuro, and hepatoprotective properties. Red beetroot (Beta vulgaris) contains red (betacyanins) and yellow (betaxanthins) pigments known as betalains. Betanin makes up 75-95% of the total betacyanins, possessed a wide range of favorable biological effects such as chemopreventive, anticarcinogenic, anti-tumorogenic, antiangiogenic, and proapoptotic effects. Methods: Red beetroot hydro-alcoholic extract and betanin were used to treat Caco-2 and HT-29 colorectal cancer cells, as well as KDR/293 normal epithelial cells. The half-maximal inhibitory concentration (IC50) was determined by prescreening MTT tests in the range of 20 to 140 µg/ml at 24 and 48 h. The cytotoxicity and apoptosis-inducing evaluations were performed via MTT assay, DAPI staining, and FACS-flow cytometry tests using determined times and doses. Moreover, the expression level of six important genes involving in the apoptosis pathway (Bcl-2, BAD, Caspase-3, Caspase-8, Caspase-9, and Fas-R) were determined using the real-time polymerase chain reaction (RT-PCR) method.Results: The IC50 doses for HT-29 and Caco-2 cell lines were determined to be about 92 μg/mL, 107 μg/mL for beetroot hydro-alcoholic extract, and 64 μg/mL, 90 μg/mL for betanin at 48 h, respectively. Our findings showed that beetroot extract and betanin significantly inhibit the growth of HT-29 and Caco-2 cell lines, time and dose-dependently, without considerable adverse effects on KDR/293 normal cells. Moreover, DAPI staining and flow cytometry results revealed significant apoptosis symptoms in treated cancerous cell lines. The expression level of pro-apoptotic genes involved in intrinsic and extrinsic apoptosis pathways (BAD, Caspase-3, Caspase-8, Caspase-9, and Fas-R) in treated HT-29 and Caco-2 cells was higher than untreated and normal cells, whereas the anti-apoptotic gene (Bcl-2) was downregulated. Conclusion: Beetroot hydro-alcoholic extract and betanin significantly inhibited cell proliferation and induced cell apoptosis (intrinsic and extrinsic pathways) via modification of effective genes in both colorectal cancer cell lines with no significant cytotoxic effects on KDR/293 normal cells. The mechanism of the anticancer effects of red beetroot extract and betanin needs to be further studied.

scholarly journals Combination of ω-3 Polyunsaturated Fatty Acids with 5-FU Inhibits SGC7901 Gastric Cancer Cell Growth via Rho-ROCK1 Signaling Pathway in Nude Mice

2021 ◽  
Author(s):  
Jiang Yiyan ◽  
Wang Keke ◽  
Lou Zhefeng ◽  
Hong Dan ◽  
Min Tao
Keyword(s):  
Gastric Cancer ◽  
Western Blot ◽  
Cancer Cells ◽  
Nude Mice ◽  
Protein Level ◽  
Combination Group ◽  
Control Group ◽  
Mrna Levels ◽  
Caspase 9 ◽  
Gastric Cancer Cells

Abstract Background: Gastric cancer is one of the most common malignancy with high mortality rate in the world. Systemic chemotherapy is thought to be an important treatment. However, due to the unsatisfactory efficiency and obvious side effects, it is urgent to detect new therapy strategy for gastric cancer. This study was aimed to investigate the effects and mechanisms of ω-3 polyunsaturated acids (PUFAs) combined with 5-FU on the growth of gastric cancer cells in nude mice. Methods: BALB/C nude mice were injected subcutaneously with SGC7901 gastric cancer cells to establish a tumor-bearing mouse model. The tumor growth in vivo was observed. Morphological of tumor specimens was observed by HE staining. The mRNA levels of RhoA, RhoC and ROCK1 in tumor tissues were detected by qPCR, and their protein levels were detected by immunofluorescence and Western Blot. Meanwhile, apoptosis –related proteins were also determined by Western Blot.Results: Compared with the NC control group, the tumor volume and weight in ω-3 PUFAs and 5-Fu groups were insignificantly lower, but significantly lower in the combination group. Compared with the abundant blood supply in the NC group, HE staining showed multifocal tumor necrosis in the three intervention groups, and this change was the most prominent in the combination group. And qPCR results showed that the mRNA levels of RhoA in the combination groups were significantly lower than this in the other groups. Immunofluorescence showed that the level of RhoA protein in the three intervention groups decline in varying degrees, especially in the combination group. Western Blot showed that the protein level of RhoA in the three intervention groups were significantly lower than those in the NC control group, especially in the combination group. Meanwhile, the protein level of ROCK1 in both 5-FU group and the combination group were significantly lower, especially in the combination group. Compared with the control group, the levels of Bcl-2 and Caspase-9 decreased in the combination group, the level of cleaved Parp was increased at the same time.Conclusion: ω-3 PUFAs combined with 5-FU may inhibit tumor growth through the Rho/ROCK pathway and promote apoptosis by down-regulating the levels of Bcl-2 and Caspase-9 and induce the increase of cleaved Parp level.

scholarly journals Silica nanoparticles inducing the apoptosis of spermatocyte cell through microRNA-450b-3p targeting MTCH2-mediating mitochondrial signaling pathway

2020 ◽  
Author(s):  
Guiqing Zhou ◽  
Jianhui Liu ◽  
Xiangyang Li ◽  
Yujian Sang ◽  
Yue Zhang ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Silica Nanoparticles ◽  
Caspase 3 ◽  
Reproductive Toxicity ◽  
Control Group ◽  
Caspase 9 ◽  
Protein Expressions ◽  
Underlying Mechanisms

Abstract Background: Silica nanoparticles (SiNPs) are found in environmental particulate matter and are proven to have adverse effects on fertility. The relationship and underlying mechanisms between miRNAs and apoptosis induced by SiNPs during spermatogenesis is currently ambiguous. Experimental design: The present study was designed to investigate the role of miRNA-450b-3p in the reproductive toxicity caused by SiNPs. In vivo, 40 male mice were randomly divided into control and SiNPs groups, 20 per group. The mice in the SiNPs group were administrated 20 mg/kg SiNPs by tracheal perfusion once every 5 days, for 35 days, and the control group were given the equivalent of a normal luminal saline. In vitro, spermatocyte cells were divided into 0 and 5 μg/mL SiNPs groups, after passaged for 30 generations, the GC-2spd cells in 5 μg/mL SiNPs groups were transfected with miRNA-450b-3p and its mimic and inhibitor. Results: In vivo, the results showed that SiNPs damaged tissue structures of testis, decreased the quantity and quality of the sperm, reduced the expression of miR-450b-3p, and increased the protein expressions of the MTCH2, BID, BAX, Cytochrome C, Caspase-9, and Caspase-3 in the testis. In vitro, SiNPs obviously repressed the viability and increased the LDH level and apoptosis rate, decreased the levels of the miR-450b-3p, significantly enhanced the protein expressions of the MTCH2, BID, BAX, Cytochrome C, Caspase-9, Caspase-3; while the mimic of miR-450b-3p reversed the changes induced by SiNPs, but inhibitor further promoted the effects induced by SiNPs.Conclusion: The result suggested that SiNPs could induce the spermatocyte apoptosis by inhibiting the miR-450b-3p expression to target promoting the MTCH2 resulting in activating mitochondrial apoptotic signaling pathways in the spermatocyte cells.

scholarly journals Ling-Gui-Zhu-Gan Decoction Protects H9c2 Cells against H2O2-Induced Oxidative Injury via Regulation of the Nrf2/Keap1/HO-1 Signaling Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Xiang Wang ◽  
Tongjuan Tang ◽  
Mengting Zhai ◽  
Ruirui Ge ◽  
Liang Wang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heart Failure ◽  
Stress Response ◽  
Caspase 3 ◽  
Underlying Mechanism ◽  
Oxidative Stress Response ◽  
Control Group ◽  
H9c2 Cells ◽  
Model Group ◽  
Blank Control Group

Objectives. Ling-Gui-Zhu-Gan decoction (LGZGD) is a potentially effective treatment for heart failure, and it showed significant anti-inflammatory potential in our previous studies. However, its ability to ameliorate heart failure through regulation of oxidative stress response is still unknown. This study was aimed to investigate the protective effect of LGZGD-containing serum on H2O2-induced oxidative injury in H9c2 cells and explore the underlying mechanism. Methods. Eighteen rats were randomly divided into two groups: the blank control group and LGZGD group. The LGZGD group rats were administrated with 8.4 g/kg/d LGZGD for seven consecutive days through gavage, while the blank control group rats were given an equal volume of saline. The serum was extracted from all the rats. To investigate the efficacy and the underlying mechanism of LGZGD, we categorized the H9c2 cells into groups: the control group, model group, normal serum control (NSC) group, LGZGD group, LGZGD + all-trans-retinoic acid (ATRA) group, and ATRA group. Malonedialdehyde (MDA) and superoxide dismutase (SOD) were used as markers for oxidative stress. Dichlorodihydrofluorescin diacetate (DCFH-DA) staining was used to measure the levels of reactive oxygen species (ROS). The apoptosis rate was detected using flow cytometry. The expression levels of pro-caspase-3, cleaved-caspase-3, Bcl-2, Bax, Keap1, Nrf2, and HO-1 were measured using western blotting. The mRNA levels of Keap1, Nrf2, and HO-1 were measured using RT-qPCR. Results. The LGZGD attenuated injury to H9c2 cells and reduced the apoptosis rate. It was also found to upregulate the SOD activity and suppress the formation of MDA and ROS. The expression levels of pro-caspase-3 and Bcl-2 were significantly increased, while those of cleaved-caspase-3 and Bax were decreased in the LGZGD group compared with the model group. As compared with the model group, the LGZGD group demonstrated decreased Keap1 protein expression and significantly increased Nrf2 nuclear expression and Nrf2-mediated transcriptional activity. ATRA was found to reverse the LGZGD-mediated antioxidative and antiapoptotic effect on injured H9c2 cells induced by H2O2. Conclusion. Our results demonstrated that LGZGD attenuated the H2O2-induced injury to H9c2 cells by inhibiting oxidative stress and apoptosis via the Nrf2/Keap1/HO-1 pathway. These observations suggest that LGZGD might prevent and treat heart failure through regulation of the oxidative stress response.

scholarly journals β-arrestin-2 up-regulates toll-like receptor 2 signaling and inhibits apoptosis in human endometrial cancer heterotransplants in nude mice

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Fanling Hong ◽  
Yujun Zhang ◽  
Wenjin Cheng ◽  
Xiuli Sun ◽  
Jianliu Wang
Keyword(s):  
Inflammatory Cytokines ◽  
Nude Mice ◽  
Caspase 3 ◽  
Glycogen Synthase ◽  
Inflammatory Factors ◽  
Toll Like Receptor ◽  
Caspase 9 ◽  
Serine Threonine Kinase ◽  
Tnf Α ◽  
Toll Like Receptor 2

Abstract Background β-arrestin-2(Arr2) functions as an anti-apoptotic factor and affects cell proliferation, but its downstream molecular pathway in endometrial carcinoma (EC) is still unclear. This study aimed to investigate the effects of the stable overexpression of Arr2 on the proliferation and apoptosis of human EC heterotransplants and the expression of associated molecules, including Toll-like receptor 2(TLR2), serine-threonine kinase Akt (Akt), glycogen synthase kinase-3β(GSK3β) and some typical inflammatory cytokines such as NF-κB p56, TNF-α and IL-6 & IL-8. Methods Human EC cell line Ishikawa, stably transfected with Arr2 full-length plasmid, was injected subcutaneously into nude mice. They were treated with 0, 10, 20 mg/kg paclitaxel and the volume and weight of the tumor tissue were measured and calculated. The necrotic index were assessed by H&E staining and microscopic observation. The levels of caspase-3, caspase-9, TLR2, NF-κB p56, Akt, GSK3β were measured by western blot, and the levels of TNF-α, IL-6, IL-8 were measured by real-time PCR. Results We found that Arr2 overexpression promoted the growth of human EC heterotransplants. Arr2 attenuated the promotion of caspase-3 and caspase-9 by paclitaxel and mediated the increase of TLR2 and several inflammatory cytokines. The levels of Akt and GSK3β were not affected. Conclusion Arr2 overexpression was associated with the increase of TLR2 and several inflammatory factors, meanwhile inhibited paclitaxel-induced anti-tumor effect on human EC heterotransplants.

Intracellular NAD+ Depletion Enhances Bortezomib-Induced Myeloma Cytotoxicity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 330-330
Author(s):  
Antonia Cagnetta ◽  
Michele Cea ◽  
Chirag Acharya ◽  
Teresa Calimeri ◽  
Yu-Tzu Tai ◽  
...  
Keyword(s):  
Cell Death ◽  
Synergistic Effect ◽  
Cell Lines ◽  
Caspase 3 ◽  
Statistical Significance ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Nicotinamide Phosphoribosyltransferase

Abstract Abstract 330 Background: Our previous study demonstrated that inhibition of nicotinamide phosphoribosyltransferase (Nampt) acts by severely depleting intracellular NAD+ content and thus eliciting mitochondrial dysfunction and autophagic MM cell death. The proteasome inhibitor Bortezomib induces anti-MM activity by affecting a variety of signaling pathways. However, as with other agents, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we demonstrate that combining Nampt inhibitor and bortezomb induces synergistic anti-MM cell death both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. Material and Methods: We utilized MM.1S, MM.1R, RPMI-8226, and U266 human MM cell lines, as well as purified tumor cells from patients relapsing after prior therapies. Cell viability and apoptosis assays were performed using Annexin V/PI staining. Intracellular NAD+ level and proteasome activity were quantified after 12, 24, and 48h exposure to single/combination drugs by specific assays. In vitro angiogenesis was assessed by Matrigel capillary-like tube structure formation assay. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, and tubulin. CB-17 SCID male mice (n = 28; 7 mice/EA group) were subcutaneously inoculated with 5.0 × 106 MM.1S cells in 100 microliters of serum free RPMI-1640 medium. When tumors were measurable (3 weeks after MM cell injection), mice were treated for three weeks with vehicle alone, FK866 (30mg/kg 4 days weekly), Bortezomib (0.5 mg/kg twice weekly), or FK866 (30 mg/kg) plus Bortezomib (0.5 mg/kg). Statistical significance of differences observed in FK866, Bortezomib or combination-treated mice was determined using a Student t test. Isobologram analysis was performed using “CalcuSyn” software program. A combination index < 1.0 indicates synergism. Results/Discussion: Combining FK866 and Bortezomib induces synergistic anti-MM activity in vitro against MM cell lines (P<0.005, CI < 1) or patient CD138-positive MM cells (P< 0.004). FK866 plus Bortezomib-induced synergistic effect is associated with: 1)activation of caspase-8, caspase-9, caspase-3, and PARP; 2) improved intracellular NAD+ dissipation; 3) suppression of chymotrypsin-like, caspase-like, and trypsin-like proteolytic activities; 4) inhibition of NF-kappa B signaling; and 5) inhibition of angiogenesis. Importantly, the ectopic overexpression of Nampt rescues this observed synergistic effect; conversely, Nampt knockdown by RNAi significantly enhances the anti-MM effect of bortezomib. In the murine xenograft MM model, low dose combination FK866 (30 mg/kg) and Bortezomib (0.5 mg/kg) is well tolerated, significantly inhibits tumor growth (P < 0.001), and prolongs host survival (2–2.5 months in mice receiving combined drugs, P = 0.001). These findings demonstrate that intracellular NAD+ levels represent a major determinant in the ability of bortezomib to induce apoptosis of MM cells, providing the rationale for clinical protocols evaluating FK866 together with Bortezomib to improve patient outcome in MM. Disclosures: Munshi: Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

High Trip13 and Low P31 Comet Cause Drug Resistance and Poor Prognosis In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3820-3820
Author(s):  
Yi Tao ◽  
Zhimin Gu ◽  
Ye Yang ◽  
Hongwei Xu ◽  
Xiaojing Hu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Caspase 3 ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Newly Diagnosed ◽  
Caspase 9 ◽  
Over Expression

Abstract Background We have recently established that increased chromosomal instability (CIN) signature is linked to drug resistance and poor outcome in multiple myeloma (MM) and other cancers. Thyroid Hormone Receptor Interactor 13 (Trip13), one of the 56 drug-resistant genes, plays a key role in chromosomal recombination and structure development during meiosis and has been reported to be increased in some malignancies including lung cancer, prostate cancer and breast cancer. In this study, we investigated how important Trip13 is in myelomagenesis and progression. Materials and Methods Gene expression profiling (GEP) was analyzed on plasma cells from 22 healthy donors, 44 patients with monoclonal gammopathy of undetermined significance (MGUS), 351 patients with newly diagnosed multiple myeloma, and 9 human myeloma cell lines, as well as on 36 sequential samples at diagnosis, pre-1st, pre-2nd and post-2nd autologous stem cell transplantation (ASCT). Over-expression and knock-down experiments of Trip13 were performed on myeloma cell lines by lentivirus transfection. Cell viability was assessed by trypan exclusion assay. Western blots were used to detect the expression of Trip13, P31 comet, caspase-8, caspase-9, caspase-3 and PARP, and checkpoint related proteins MAD2 and CDC20 in Trip13 overexpressed or Trip13 shRNA-transfected myeloma cells. Results Sequential GEP samples showed that Trip13 expression increased in 8 of 9 patients after chemotherapy and ASCT compared to the samples at diagnosis strongly suggesting that increased Trip13 is associated with drug resistance. Trip13 was already significantly increased in MGUS patients, newly diagnosed MM patients and MM cell lines compared with normal plasma cells. Furthermore, Trip13 was significantly higher in high-risk MMs than in low-risk MMs and increased Trip13 was linked to an inferior event-free survival (EFS) (p<0.01) and overall survival (OS) (p<0.01) in 351 newly diagnosed MMs. In contrast, the Trip13-interacting gene P31 comet was down-regulated in high-risk MMs and high expression of P31 was associated with good outcome. Interestingly, patients with high Trip13 and low P31 comet have the worst outcome compared to patients with only one of these, suggesting the interaction of Trip 13 and p31 has a synergistic effect on MM progression. Transfection of Trip13 into ARP1 and OCI-My5 cells significantly increased cell proliferation, while knock-down Trip13 in OCI-My5, H929, RPMI8226 cells inhibited cell growth and induced MM cell apoptosis with increases of cleaved caspase-8, caspase-9, caspase-3 and PARP. Mechanistic studies showed that Trip13 over-expression decreased P31comet and MAD2 expression by western blotting, but increased CDC20. Conclusions The association of increased Trip13 and decreased p31 is a good biomarker for MM drug resistance and poor prognosis. Our results also show Trip13 and P31 comet could be potential targets to overcome drug resistance in MM. Disclosures: No relevant conflicts of interest to declare.

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