scholarly journals Inhibition of autophagy enhanced chemosensitivity in cisplatin resistant hypopharyngeal squamous carcinoma cells

2020 ◽  
Author(s):  
pin dong ◽  
Jia Zhang ◽  
Wei Mao ◽  
Yuying Liu ◽  
Jian Ding ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multidrug Resistance ◽  
Survival Rate ◽  
Western Blot ◽  
Cell Line ◽  
Cell Counting ◽  
G1 Arrest ◽  
Fadu Cell ◽  
Squamous Carcinoma Cells ◽  
Fadu Cells

Abstract Background: Hypopharyngeal carcinoma is characterized by high degree of malignancy. The most common pathological type is squamous cell carcinoma (HSCC). It has been confirmed that high autophagy level promotes the development of hypopharyngeal cancer in recent years. Clinical researches have reported that high autophagy level often caused insensitivity to chemotherapy, a common phenomenon that greatly reduces therapeutic effect in cisplatin-resistant tumor cell lines. Therefore, exploring internal mechanisms of autophagy on cisplatin resistant HSCC is necessary for founding theoretical basis for synergistic antitumor drugs by interfering with autophagy.Methods: Part I: Cisplatin-resistant FaDu cell line was established and cultured. Cell counting kit-8 was used to detect drug resistance. Inverted microscope was used to observe the morphological changes at different concentrations, then the survival rate was calculated. After MDC staining, the autophagic vacuoles were observed by fluorescence microscopy. The expression of Beclin1 from each group was confirmed by RT-PCR and Western blot method. Part II: Beclin1 was knocked down by plasmid transfection, autophagy inhibitor 3-MA was applied for cisplatin-resistant cells intervention. Cell cycle was detected using flow cytometry assay, apoptosis with necrosis was detected by staining with propidium iodide (PI). CCK-8 was used to observe the cell survival rate in each group. The expression of autophagy-related gene Beclin1,LC3I,LC3II,Atg-5 and P62 in each group was verified by Western blot analysis.Results: Cisplatin-resistant FaDu cell line can be stably constructed by cisplatin intervention. Compared with normal group, autophagy and its related protein Beclin1 expression was enhanced in cisplatin resistant FaDu cells. Autophagy inhibition group showed significant cell cycle changes, mainly manifested by G1 arrest, increased apoptosis rate and significantly decreased survival rate at 24h level. Furthermore, Western blot showed that expression of Beclin1, lc3i, lc3ii, atg-5 protein decreased significantly after the inhibitor used, while the expression of p62 up-regulated, which also confirmed autophagy flow was blocked.Conclusion: Our work confirmed high autophagy level is important for the cisplatin-resistance of HSCC and insensitivity to chemotherapy. The use of 3-MA and Beclin 1 inhibition can significantly reduce autophagy level of cisplatin-resistant FaDu cells, arresting its cell cycle, promote apoptosis and reverse the multidrug resistance condition. These results provide the experimental basis for overcoming multidrug resistance through combination chemotherapy.#These authors contributed equally to this work.

3-MA Enhanced Chemosensitivity in Cisplatin Resistant Hypopharyngeal Squamous Carcinoma Cells via Inhibiting Beclin -1 Mediated Autophagy

2020 ◽  
Vol 26 ◽  
Author(s):  
Jia Zhang ◽  
Wei Mao ◽  
Yuying Liu ◽  
Jian Ding ◽  
Jie Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Squamous Cell Carcinoma ◽  
Survival Rate ◽  
Western Blot ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Cisplatin Resistance ◽  
Carcinoma Cells ◽  
Fadu Cell ◽  
Fadu Cells

Background: Hypopharyngeal carcinoma is characterized by high degree of malignancy. The most common pathological type is squamous cell carcinoma (HSCC). Cisplatin (cis-diamminedichloroplatinum, CDDP) is one of the most widely used chemotherapeutic drugs nowadays and cisplatin resistance is a major problem in current treatment strategies. Clinical researches have reported that high autophagy level often caused insensitivity to chemotherapy, a common phenomenon that greatly reduces therapeutic effect in cisplatin-resistant tumor cell lines. 3-methyladenine (3-MA), an inhibitor of PI3K, plays a vital role in the formation and development of autophagosomes. Therefore, we speculate that the use of 3-MA may reduce cisplatin resistance in hypopharyngeal squamous cell carcinoma (HSCC). Methods: Part I: Cisplatin-resistant FaDu cell line (Human hypopharyngeal squamous cell carcinoma cells) was established and cultured. Cell counting kit-8 was used to detect drug resistance. Inverted microscope was used to observe the morphological changes at different concentrations, then the survival rate was calculated. After MDC staining, the autophagic vacuoles were observed by fluorescence microscopy. The expression of Beclin1 from each group was confirmed by RTPCR and Western blot method. Part II: 3-MA was applied for cisplatin-resistant cells intervention, Beclin1 was knocked down by plasmid transfection. Cell cycle was detected using flow cytometry assay, apoptosis with necrosis was detected by staining with propidium iodide (PI). CCK-8 was used to observe the cell survival rate in each group. The expression of autophagy-related protein Beclin1, LC3I, LC3II, Atg-5 and P62 in each group was verified by Western blot analysis. Results: Cisplatin-resistant FaDu cell line can be stably constructed by cisplatin intervention. Compared with normal group, autophagy and its related protein Beclin1 expression was enhanced in cisplatin resistant FaDu cells. Autophagy inhibition group showed significant cell cycle changes, mainly manifested by G1 arrest, increased apoptosis rate and significantly decreased survival rate at 24h level. The number of autophagy vacuoles were significantly reduced in the 3-MA group. Furthermore, Western blot showed that expression of Beclin1, lc3-I, lc3-II, atg-5 protein decreased significantly after 3-MA intervention, while the expression of p62 up-regulated, which also confirmed autophagy flow was blocked. Conclusion: Our work confirmed that enhanced autophagy is an important cause of cisplatin resistance in FaDu cells. The use of 3-MA can significantly reduce autophagy level and arresting its cell cycle, promote apoptosis and reverse the cisplatin resistance condition, this effect is partly mediated by inhibition of Beclin-1 expression. Our data provides a theoretical basis for the application of 3-MA in overcoming cisplatin resistance in hypopharyngeal cancer.

Identification and Expression Analysis of CD73 Inhibitors in Cervical Cancer

2020 ◽  
Vol 16 ◽  
Author(s):  
Jamshed Iqbal ◽  
Ayesha Basharat ◽  
Sehrish Bano ◽  
Syed Mobasher Ali Abid ◽  
Julie Pelletier ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Western Blot ◽  
Hela Cell ◽  
Cell Line ◽  
Cell Apoptosis ◽  
Immunofluorescence Staining ◽  
Cell Cycle Analysis ◽  
Hela Cell Line ◽  
Cell Line Hela

Aims: The present study was conducted to examine the inhibitory effects of synthesized sulfonylhydrazones on the expression of CD73 (ecto-5′-NT). Background: CD73 (ecto-5′-NT) represents the most significant class of ecto-nucleotidases which are mainly responsible for dephosphorylation of adenosine monophosphate to adenosine. Inhibition of CD73 played an important role in the treatment of cancer, autoimmune disorders, precancerous syndromes, and some other diseases associated with CD73 activity. Objective: Keeping in view the significance of CD73 inhibitor in the treatment of cervical cancer, a series of sulfonylhydrazones (3a-3i) derivatives synthesized from 3-formylchromones were evaluated. Methods: All sulfonylhydrazones (3a-3i) were evaluated for their inhibitory activity towards CD73 (ecto-5′-NT) by the malachite green assay and their cytotoxic effect was investigated on HeLa cell line using MTT assay. Secondly, most potent compound was selected for cell apoptosis, immunofluorescence staining and cell cycle analysis. After that, CD73 mRNA and protein expression were analyzed by real-time PCR and Western blot. Results: Among all compounds, 3h, 3e, 3b, and 3c were found the most active against rat-ecto-5′-NT (CD73) enzyme with IC50 (µM) values of 0.70 ± 0.06 µM, 0.87 ± 0.05 µM, 0.39 ± 0.02 µM and 0.33 ± 0.03 µM, respectively. These derivatives were further evaluated for their cytotoxic potential against cancer cell line (HeLa). Compound 3h and 3c showed the cytotoxicity at IC50 value of 30.20 ± 3.11 µM and 86.02 ± 7.11 µM, respectively. Furthermore, compound 3h was selected for cell apoptosis, immunofluorescence staining and cell cycle analysis which showed promising apoptotic effect in HeLa cells. Additionally, compound 3h was further investigated for its effect on expression of CD73 using qRT-PCR and western blot. Conclusion: Among all synthesized compounds (3a-3i), Compound 3h (E)-N'-((6-ethyl-4-oxo-4H-chromen-3-yl) methylene)-4-methylbenzenesulfonohydrazide was identified as most potent compound. Additional expression studies conducted on HeLa cell line proved that this compound successfully decreased the expression level of CD73 and thus inhibiting the growth and proliferation of cancer cells.

Induction of G1 Arrest and Apoptosis in a Conditional Expression Model of AML1/ETO: P53-Independent Upregulation of P21/WAF/CIP1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2576-2576
Author(s):  
Tobias Berg ◽  
Manfred Fliegauf ◽  
Jurij Pitako ◽  
Jan Burger ◽  
Mahmoud Abdelkarim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Parp Cleavage ◽  
G1 Arrest ◽  
U937 Cells ◽  
Negative Cell ◽  
Conditional Expression

Abstract Background: The translocation (8;21) is the most common chromosomal rearrangement in AML, resulting in the expression of the fusion protein AML1/ETO. We have developed an ecdysone-inducible U937 model, in which AML1/ETO is expressed in response to treatment with Ponasterone (Pon) A (Fliegauf et al, Oncogene 2004). This model system was used to determine the cellular effects of AML1/ETO and to identify its target genes in U937 cells. Methods: Effects of AML1/ETO expression upon cell growth, viability, cell cycle and apoptosis were analyzed by trypan blue exclusion, FACS analysis using propidium iodide and DiOC6 staining, DNA laddering and Western blot for PARP cleavage, respectively. The gene expression profile of U937 with and without conditional AML1/ETO expression was assessed using Affymetrix U133A microarrays. Wild-type U937 cells with and without PonA treatment as well as AML1/ETO-negative and AML1/ETO-positive myeloid cell lines served as controls. Northern and Western Blotting were used for validation of expression changes. Results: Induction of AML1/ETO expression in U937 resulted in reduced cell growth, G1 arrest and in apoptosis beginning 48–72 hours after PonA treatment. To investigate the underlying mechanisms, microarray analysis was performed. Expression profiles of AML1/ETO-positive and AML1/ETO-negative cell lines formed distinct clusters. Based on stringent criteria, 191 different genes were found upregulated, whereas 37 were downregulated upon expression of AML1/ETO in U937. The identified genes were screened for genes with known functions in cell cycle and apoptosis by automated and manual review and included 13 apoptosis-related genes. Among them, the CDK inhibitor p21/WAF/CIP1 was upregulated 19-fold upon induction of AML1/ETO, whereas the apoptosis regulator MCL-1 was induced 2.5-fold. Based on our criteria, no differential expression of other transcriptionally-controlled apoptosis regulators (such as BCL2, BAX, BAK1, BAD or c-flip) was noted. Northern and Western Blot analysis confirmed the strong induction of p21/WAF/CIP1 that paralleled the expression of AML1/ETO 10 hours after PonA treatment. Induction of p21/WAF/CIP1 was independent of the tumor suppressor protein p53 (Dou et al., Proc. Natl. Acad. Sci. 1995), and by Western blot, p53 was undetectable in U937. Northern Blot analysis revealed a higher expression of p21/WAF/CIP1 in the AML1/ETO-positive cell lines Kasumi-1 and SKNO-1 than in the AML1/ETO-negative cell lines HL-60, KG-1 and U937, supporting our finding that AML1/ETO may induce p21/WAF/CIP1. Conclusions: AML1/ETO expression resulted in increased expression of p21/WAF/CIP1, which might contribute to the observed growth arrest and induction of apoptosis caused by the conditional expression of AML1/ETO.

scholarly journals The Antitumor Activity of Pseudoginsengenin DQ Against Hypopharyngeal Cancer Cells via Targeting HIF-1α-GLUT1 Pathway

2021 ◽  
Author(s):  
Sanchun Wang ◽  
Yu Cai ◽  
Qingjie Feng ◽  
Jing Gao ◽  
Bo Teng
Keyword(s):  
Cell Cycle ◽  
Molecular Docking ◽  
Antitumor Activity ◽  
Glucose Uptake ◽  
Cancer Cells ◽  
Docking Study ◽  
Hypopharyngeal Cancer ◽  
Cell Counting ◽  
Molecular Docking Study ◽  
Fadu Cells

Abstract Background The ginsenosides have been reported to possess a variety of biological activities. Synthesized from the ginsenoside Protopanaxadiol (PPD), the octanone Pseudoginsengenin DQ (PDQ) may have stronger pharmacological effects as a secondary ginsenoside. Nevertheless, its antitumor activity and molecular mechanism against hypopharyngeal cancer cells remains unclear. Methods Cell Counting Kit-8, cell cycle assay and cell apoptosis assay were conducted to detect FADU cells proliferation, cell phase and apoptosis. The interactions between PDQ and HIF-1α were investigated by a molecular docking study. The expression of HIF-1α, GLUT1, apoptosis related proteins was tested by western blotting, direct stochastic optical reconstruction microscopy (dSTORM) and qRT-PCR. Glucose uptake assay was used to assess the glucose uptake capacity of FADU cells. Results PDQ was found to suppress the proliferation, reduce glucose uptake, induce the cell cycle arrest and apoptosis of FaDu cells. Molecular docking study demonstrated that PDQ could interact with the active site of HIF-1α. PDQ decreased the expression and mRNA levels of HIF-1α and its downstream factor GLUT1. Moreover, dSTORM results showed that PDQ reduced GLUT1 expression on the cell membrane but also inhibited its clustering. Conclusion Our work elucidated that the antitumor effect of PDQ is related to its downregulation of HIF-1α-GLUT1 pathway, suggesting that PDQ could be a potential therapeutic agent for hypopharyngeal cancer treatment.

scholarly journals Chidamide, a Novel Histone Deacetylase Inhibitor, Displays Potent Antitumor Activity Against MDS Cells Mainly through JAK2/STAT3 Signaling Inhibition

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5233-5233 ◽  
Author(s):  
Chunkang Chang ◽  
Sida Zhao ◽  
Juan Guo ◽  
Youshan Zhao ◽  
Chengming Fei ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Histone Deacetylases ◽  
Conflicts Of Interest ◽  
Histone H3 ◽  
Cell Counting ◽  
Dependent Manner ◽  
Cytotoxic Effects ◽  
Hdac Activity ◽  
Stat3 Signaling

Abstract Background: Many studies have indicated that histone deacetylases (HDAC) activity is always increased in a lot of human tumors, and inhibition of HDAC activity are promising new strategies in the treatment of cancers. Chidamide (CS055/HBI-8000), a novel histone deacetylases inhibitor (HDACi) of the benzamide class, is currently under clinical trials. Despite emerging information on the effect of chidamide in multiple cancers, little is yet known about mechanism of action, and there were few reports about this drug's effects on myelodysplastic syndromes (MDS). In this study, we aimed to investigate the antitumor activities of chidamide on MDS cell line SKM-1 and to explore the possible mechanism. Methods: We treated MDS cells with different concentrations of chidamide. The effect of chidamide on HDAC activity of MDS cells was studied by using a HDAC Fluorometric Activity Assay Kit. The induction of histone acetylation was confirmed by detecting acetylated histone H3 and H4 using Western blot analysis. The effect of chidamide on the proliferation of MDS cells was analyzed by Cell Counting Kit (CCK-8) assay. Apoptosis were detected by Annexin V/propidium iodide (PI) double-labeled cytometry. Cell cycle was analyzed by a PI method. The acetylation levels of genes promoterassociated histone H3 and H4 were examined by chromatin immunoprecipitation (ChIP) and quantitative real-time PCR. Quantitativereal-time PCR and Western blot were used to detect the expression of signaling pathway factors (SOCS3, JAK2, STAT3,Bcl-XL,Bcl-2 and Mcl-1). Results: Our results demonstrated chidamide suppressed MDS cells growth in a time- and dose-dependent manner, and induced cell apoptosis and cell cycle arrest at G0/G1phase. Chidamide was able to significantly inhibit the HDAC activity and increase the acetylated histoneH3 and H4 of MDS cells. ChIP analysis further indicated that chidamide induced acetylated histones accumulation in the promoter of SOCS3.Moreover, chidamide upregulated the mRNA and protein expression ofSOCS3, and also significantly downregulated JAK2/STAT3 signaling. Further, we identified reduced expression of STAT3 downstream targets with treatment of chidamide, including Bcl-XL, Bcl-2 and Mcl-1, which may explain the cytotoxic effects of chidamide on MDS cells. Conclusion: These results demonstrate that chidamide possesses significant cytotoxic effects on MDS cells mainly through histone modifications of SOCS3 and consequently JAK2/STAT3 signaling inhibition. Therefore, Our data provide rationale for clinical investigation of chidamide in MDS. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effect of DMSA Modified Fe3O4 and Multidrug Resistance Modulator HZ08 on the Reversal of Multidrug Resistance in K562/A02 Cell Line

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5235-5235
Author(s):  
Fei Shen ◽  
Mingfei Zhou ◽  
Xuzhang Lu ◽  
Yanping Chen ◽  
Baoan Chen
Keyword(s):  
Multidrug Resistance ◽  
Cell Line ◽  
Real Time ◽  
Caspase 3 ◽  
Conflicts Of Interest ◽  
Morphological Changes ◽  
Leukemia Cell ◽  
Cell Counting ◽  
Leukemia Cell Line ◽  
Gene And Protein Expression

Abstract The objective of the present study was to investigate the reversible effect of HZ08 and daunorubicin(DNR) combined with dimercaptosuccinic acid modified iron oxide (DMSA-Fe3O4) magnetic nanoparticles (MNPs) in human chronic leukemia cell line K562/A02, and the mechanism potentially involved. The growth inhibition rate of K562/A02 cells was determined by Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis and intracellular concentration of DNR were detected by flow cytometry (FCM). DAPI staining was used to view apoptotic cellular morphology. Subsequently, transcription levels of MDR1 mRNA and expression levels of P-glycoprotein (P-gp) and caspase-3 were determined by real time polymerase chain reaction (real-time PCR) and Western blotting analysis, respectively. group clearly exhibited more morphological changes (severe structural alterations) than other groups. In addition, transcription of MDR1 gene and protein expression of P-gp and caspase-3 of K562/A02 cells were regulated at the most remarkable extent in DNR-HZ08-MNPs group when compared with other groups. These findings suggest that the remarkable effects of the novel DNR-HZ08-MNPs on multidrug resistant K562/A02 leukemia cells would be a promising strategy for overcoming multidrug resistance. Disclosures No relevant conflicts of interest to declare.

129 microRNA-183~96~182 CLUSTER PROMOTE BOVINE GRANULOSA CELL PROLIFERATION THROUGH COORDINATED REGULATION OF FOXO1

2016 ◽  
Vol 28 (2) ◽  
pp. 194
Author(s):  
S. Gebremedhn ◽  
D. Salilew-Wondim ◽  
M. Hoelker ◽  
F. Rings ◽  
C. Neuhoff ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Protein Expression ◽  
Statistical Significance ◽  
Flow Cytometric Analysis ◽  
Cell Counting ◽  
G1 Arrest ◽  
Mirna Cluster ◽  
Mrna And Protein Expression ◽  
Coordinated Regulation

Among other microRNA clusters, we previously showed that the miR-183~96~182 cluster (miR-183, miR-96, and miR-182) is abundantly expressed in bovine granulosa cells (bGC) of preovulatory dominant follicles obtained at the follicular phase of the bovine oestrous cycle. Moreover, this miRNA cluster are validated to coordinately target the Fork head O1 (FOXO1), a subfamily of transcription factors that regulate genes involved in cell proliferation, apoptosis, cell cycle arrest, and metabolism. However, the functional involvement of miR-183~96~182 cluster in bGC function by regulation of FOXO1 is not yet determined. Here, we aimed to investigate the function of miR-183~96~182 cluster in bGC using in vitro cell culture model. For this, bGC were aspirated from ovarian follicles (Ø 3–5 mm) obtained from local abattoir. Cells were plated in 24-well plate (2.5 × 105 cells well–1) in DMEM/F-12 (Sigma, Germany) supplemented with 10% FBS (GIBCO, Grand Island, NY) and 1% penicillin/streptomycin (GIBCO) and incubated at 37°C in 5% CO2. Transfection of bGC with miRNA mimics, inhibitors, FOXO1-siRNA, and appropriate controls (Exiqon, Vedbæk, Denmark) was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cell proliferation was determined using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technology, Kumamoto, Japan). Cell cycle distribution was determined with flow cytometric analysis. Total RNA was isolated using miRNeasy mini kit (Qiagen, Hilden, Germany), quantification of target gene was performed using qPCR, and data were analysed using ΔΔCT method. Differences in the mean expression values between treatments were analysed with two-tailed Student’s t-test and statistical significance was defined at P ≤ 0.05. Results showed that a sponge effect was observed upon inhibition in individual miRNA of the cluster, which could be attributed to the partial sequence similarity among cluster members. Both FOXO1 mRNA and protein expression were significantly reduced upon transfection of bGC with miR-183~96~182 cluster mimics, while miR-183~96~182 cluster inhibition increased both FOXO1 mRNA and protein expression. Transfection of bGC with miR-183~96~182 mimics promoted cell proliferation, while inhibition tends to slow down proliferation. Furthermore, the proportion of bGC under G0/G1 arrest markedly declined (P < 0.05), while the S and G2/M phases increased in response to miR-183~96~182 mimicking. Selective knockdown of FOXO1 with FOXO1-siRNA significantly reduced FOXO1 mRNA and protein expression. Interestingly, knockdown of FOXO1 showed similar phenotypic effects such as that of miR-183~96~182 mimics transfection, which resulted in elevated bGC proliferation and reduction in the proportion of cells under G0/G1 arrest. In conclusion, overexpression of miR-183~96~182 cluster promote bGC proliferation and G0/G1 to S and G2/M cell cycle transition through coordinated regulation of genes in the FOXO1 signaling axis.

An Evaluation of the Cytotoxic Effect of the Natural Product Aaptamine Against t(4;11) Leukemias

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1624-1624 ◽  
Author(s):  
Chandrima Sinha ◽  
John Bowling ◽  
Aman Seth ◽  
Bensheng Ju ◽  
Bhaskar Kahali ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Treated Group ◽  
Jurkat Cells ◽  
G1 Arrest ◽  
Thp1 Cell ◽  
Rb Phosphorylation

Abstract The marine environment has been shown to be a rich source of pharmacologically-active secondary metabolites. Three marine- compounds have FDA approval for cancer indications. Aaptamine is a sponge-derived alkaloid that exhibits multiple pharmacological activities including proapoptotic/antiproliferative effects on leukemia cell lines. The effect of the aaptamine class has not been previously studied for high risk leukemias with mixed-lineage leukemia (MLL) gene rearrangements. Using the CellTiter-Glo cell viability assay we evaluated the cytotoxic effect of aaptamine against a panel of leukemia cell lines. We observed that cell lines containing t(4;11) are the most sensitive to aaptamine. Translocation (4;11) is associated with mixed-lineage leukemia and responsible for a very aggressive and refractory pediatric leukemia. Specifically, infants less than one year with t(4;11) have poor survival rates (≈ 19%) and new therapies are urgently needed. Interestingly other MLL cell lines that contain t(9;11) are comparatively less susceptible to aaptamine-mediated cytotoxicity. Jurkat cells overexpressing MLL-AF4 fusion protein are also more sensitive to aaptamine-induced cytotoxicity than wild type or MLL-AF9 overexpressing Jurkat cells indicating the specificity of aaptamine for t(4;11). To further confirm the specificity we conducted a flow based apoptosis assay and observed that aaptamine induces significant apoptosis and necrosis in RS4;11 and MV4;11 cell lines starting at 10µM but not in the t(9;11) containing THP1 cell line. We also found that aaptamine treatment induced G0/G1 arrest specifically in t(4;11) containing cell lines but not in THP1. Additionally we observed that aaptamine did not induce any resistance to the sensitive cell lines after 27 days of chronic exposure. Importantly the compound was well tolerated by healthy activated PBMCs and mice at high concentrations. In order to decipher the mechanism of specificity, we conducted a global proteomic study with treated and untreated RS4;11 and THP1 cell lines. Our proteomic data revealed a significant upregulation of p21 and p27 in aaptamine treated RS4;11 cells but not in THP1. In agreement with the proteomic data, we observed a dose-dependent upregulation of p21 and p27 in both protein and mRNA levels in RS4;11 and MV4;11 cells but not in resistant THP1 cells. Using p21 and p27 promoter-driven luciferase reporter constructs, we observed a significant upregulation of luminescence signal in the RS4;11 cell line at much lower concentration of aaptamine (1µM) whereas the THP1 cell line required 50µM of aaptamine for significant increase in luminescence signal. Cyclin-dependent kinase regulates the G1/S cell cycle transition by phosphorylating retinoblastoma protein (RB). Upregulation of cyclin-dependent kinase inhibitors, such as p27 and p21, promote RB hypophosphorylation and induce G0/G1 arrest. To confirm that this molecular mechanism is responsible for aaptamine induced G0/G1 arrest, we investigated the effect of aaptamine on Rb phosphorylation. We observed a dose dependent downregulation of Rb phosphorylation by aaptamine in sensitive cell lines and predicted it as a major cause of cell cycle arrest. Previous studies have shown that translocation (4;11) is associated with p27 upregulation; thus we hypothesize by further upregulating p27, aaptamine may induce G0/G1 arrest specifically in t(4;11) containing cell lines. To validate the efficacy of aaptamine in vivo, we xenografted 10 NSG mice with 1 million luciferase expressing RS4;11 cells. Four days after leukemia induction we treated half of the mice with subcutaneous injection of aaptamine (100mg/kg, daily) and the other half received vehicle treatment. Bioluminescence imaging (BLI) data revealed a significantly lower disease (p< 0.03) burden in the aaptamine treated group compared to vehicle treated group after 2 weeks. These findings are being confirmed in patient samples. Additional aaptamine analogs are being designed and will be evaluated for improved therapeutic efficacy. Together our in vitro and in vivo findings suggest that by inducing p21 and p27 aaptamine can induce cell cycle arrest and eventually apoptosis specifically in leukemia cells that contain t(4;11) with relatively low toxicity . Therefore the aaptamine class of drug may provide additional therapeutic options for t(4;11) containing high-risk MLL leukemia patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Regulation of MAPKs Signaling Contributes to the Growth Inhibition of 1,7-Dihydroxy-3,4-dimethoxyxanthone on Multidrug Resistance A549/Taxol Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Jian Zuo ◽  
Hui Jiang ◽  
Yan-Hong Zhu ◽  
Ya-Qin Wang ◽  
Wen Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multidrug Resistance ◽  
Western Blot ◽  
Cell Cycle Arrest ◽  
Nsclc Cell Line ◽  
Potent Inducer ◽  
Cell Lung Carcinoma ◽  
Cycle Arrest ◽  
Growth Inhibitory ◽  
Securidaca Inappendiculata

1,7-Dihydroxy-3,4-dimethoxyxanthone (XAN) is a bioactive compound isolated fromSecuridaca inappendiculataHassk. and validated with antiproliferative activities on a panel of cancer cell lines. This study was designed to investigate its growth inhibitory effects on multidrug resistance (MDR) non-small cell lung carcinoma (NSCLC) cell line A549/Taxol and explore the possible linkage between modulation of MAPKs and the bioactivities. Its growth inhibitory potency on the cells was estimated by MTT assay, and flow cytometric analysis was employed to investigate its potential cell cycle arrest and proapoptosis effects. Expressions of hallmark proteins were assessed by Western-Blot method. The results showed A549/Taxol cells were sensitive to XAN. XAN inhibited the proliferation of A549/Taxol cells in the time and concentration dependent manners. It acted as a potent inducer of apoptosis and cell cycle arrest in the cells. Western-Blot investigation validated the proapoptosis and cell cycle arrest activities of XAN and the potential of MDR reversion. Upregulation of p38 by XAN, which accounted for the cell cycle arrest at G2 phase, and the downregulation of ERK associated with the proapoptosis activity were also revealed. Further analysis found p53 may be the central role mediated the bioactivities of MAPKs in A549/Taxol cells. Based on these evidences, a conclusion has been deduced that XAN could be a potential agent for MDR NSCLC therapy targeting specifically MAPKs.

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