Elevated ANXA1 Expression Causes Glucocorticoid Resistance in Acute Lymphoblastic Leukemia Via Activating the Wnt/β-Catenin Signaling Pathway

Background: Acute lymphoblastic leukaemia (ALL) is one of the most common clonal malignant diseases in children, and it stems from unchecked proliferation of lymphoid progenitor cells. Glucocorticoids (GCs) such as prednisolone and dexamethasone are used as a chemotherapeutic drug in the treatment of ALL. GC-induced cell mortality is first mediated by the activation of glucocorticoid receptor (GR), followed by its translocation into the nucleus to activate or inhibit gene transcription. However, up to ~20% patients with leukemia relapse and become resistant to GCs. Therefore, a better understanding the molecular basis of chemoresistance in ALL would provide novel therapeutic opportunities for patients. Methods: By analyzing the published mRNA expression profiles (GSE5280; GSE94302) obtained from NCBI (https://www.ncbi.nlm.nih.gov/geo/), we found that higher expression of ANXA1 was significantly associated with decreased overall survival of ALL patients. We also examined the expression of ANXA1 at mRNA and protein levels in a variety of ALL cell lines by using qRT-PCR and western blot analyses. The mRNA and protein expression of ANXA1 in ALL cell lines and patients were determined using Real-time PCR and Western blot respectively. Functional assays, such as CCK-8, FACS, and Tunel assay used to determine the oncogenic role of ANXA1 in ALL progression. Furthermore, western blotting and luciferase assay were used to determine the mechanism of ANXA1 promotes chemoresistance in ALL cells. Results: The expression of ANXA1 was markedly upregulated in ALL cell lines and patients, and high ANXA1 expression was associated with relapsed/refractory ALL patients. ANXA1 overexpression confers glucocorticoids (GCs) resistance on ALL cells; however, down-regulated of ANXA1 sensitized ALL cell lines to GC both in vitro and in vivo. Additionally, ANXA1 upregulated the levels of FPRs by promoting Wnt/β-catenin signalling. Conclusions: Our findings provided evidence that ANXA1 is a potential therapeutic target for patients with ALL. Targeting ANXA1 signaling may be a promising strategy to enhance GC response during ALL chemo-resistance. Disclosures No relevant conflicts of interest to declare.

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Obinutuzumab (GA101) Significantly Enhances Cell Death and ADCC Compared to Rituximab Against CD20+ sensitive and Rituximab Resistant B-Cell Non-Hodgkin Lymphoma (NHL) and Lymphoblastic Leukemia (BLL)

Blood ◽

10.1182/blood.v120.21.4865.4865 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4865-4865 ◽

Cited By ~ 1

Author(s):

Aradhana Awasthi Tiwari ◽

Janet Ayello ◽

Carmella van de Ven ◽

Danielle Glassman ◽

Anthony Sabulski ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Nk Cells ◽

Cell Lines ◽

Caspase 3 ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Dismal Prognosis

Abstract Abstract 4865 Background: Patients who relapse with CD20+ B-NHL and B cell lymphoblastic leukemia (B-LL) have a dismal prognosis, often associated with chemotherapy resistance (Cairo et al. JCO, 2012,Mils/Cairo et al. BJH,2012) and often require alternative therapeutic strategies. Rituximab (RTX) in combination with FAB 96 chemotherapy is a safe, well-tolerated treatment that is associated with > 90% EFS in children with newly diagnosed and advanced mature B-Cell NHL (Cairo M.S. et al. ASCO, 2010). Resistance to RTX, however, may predispose patients with CD20+ NHL to an increase risk of relapse and or disease progression (Barth/Cairo et al. BJH, 2012; Tsai et al. Cl. Can. Res, 2012). Obinutuzumab (GA101), a novel type II glycoengineered CD20 antibody of the IgG1 isotype, mediates enhanced cell death vs RTX and has a glycoengineered Fc region that induces significantly enhanced ADCC (Mössner et al. Bld, 2010; Niederfellner G. et al. Bld, 2011; Bologna L et al. JI, 2012). Objective: To evaluate the in-vitro efficacy of GA101 compared to RTX against RTX sensitive and resistant CD20+ B-NHL and B-LL cell lines. Methods: Raji (CD20+,ATCC, Manhass, VA), U698-M (CD20+, DSMZ, Germany), Loucy cells (CD20−) (T-ALL) (ATCC, Manhass, VA) and Raji-2R and Raji-4RH (generously supplied by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS and incubated with GA101 and/or RTX at 100 μg/ml for 24 hrs (n=6), 48 and 72 hrs (n=5). Cell death was evaluated by staining with AnnexinV/7AAD and flow-cytometry. Loucy cells (CD20−) were used as the negative control. The caspase 3/7 activity was measured by FAM caspase 3/7 assay kit by FLICA™ methodology. RSCL, RRCL, U698-M and Loucy were incubated with GA101 and RTX treatment for 24, 48 and 72 hrs, and caspase3/7 activity was detected by FACS using 488 nm excitation and emission filter (n=3). ADCC were performed with K562-IL-15–41BBL expanded NK cells (Ayello/Cairo et al. ASH, 2010) as well as IL-2 expanded NK cells, at 20:1 effector: target ratio (E: T, n=3) using europium release assay (Perkin-Elmer). Results: GA101 induced significantly more cell death compared to RTX in B-NHL and BLL cell lines. (Table-1) GA101 vs RTX shows a significantly increase in caspase 3/7 activity in Raji 16.92±0.84% vs 11.76±0.08% compared to Raji2R 6.7±0.62% vs 2.8±0.7%, Raji4RH 5.8±0.35% vs 2.0±0.3% and U698-M 12.54±0.44% vs 9.6±0.95% compared to Loucy 3.22±0.45% vs 2.59±0.05%, respectively, at 24 hrs of treatment (p<0.0001). GA101 vs RTX also elicited a significant increase a ADCC with K562-IL15–41BBL expanded NK cells, in Raji 73.8±8.1% vs 56.81±4.6% compared to Raji-2R 38.0±2.0% vs 21.6±1.2%, Raji-4RH 40.0±1.6% vs 0.5±1.1% and U698-M 70.0±1.6% vs 45.56±0.1%, compared to Loucy 21.67±0.48% vs 15.92±0.52%, respectively (p<0.001) at day 7.The IL-2 alone expanded Hu-NK cells demonstrated a reduction of 10–20% cytotoxicity compared to K562-IL15–41BBL Hu-NK cells at day 7 against BLL, RSCL and RRCL, in-vitro. Conclusion: Obinutuzumab compared to RTX significantly enhanced cell death, caspase3/7 activity and NK mediated ADCC in sensitive and RTX resistant B-NHL and B-LL. Obinutuzumab represents a promising candidate for treating RTX sensitive and resistant CD20+ B-Cell Lymphomas and lymphoblastic leukemia. Further studies will investigate the combination of activated NK cells or chemotherapy that may enhance or synergize with the efficacy of GA101 (Obinutuzumab) both in -vitro and in-vivo in xenografted NOD/SCID mice. Disclosures: No relevant conflicts of interest to declare.

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ALL-Associated JAK1 Mutations Confer Hypersensitivity to the Anti-Proliferative Effect of Type I Interferon.

Blood ◽

10.1182/blood.v114.22.3066.3066 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3066-3066

Author(s):

Tekla Hornakova ◽

Sabina Chiaretti ◽

Muriel Lemaire ◽

Robin Foà ◽

Marco Tartaglia ◽

...

Keyword(s):

Cell Lines ◽

Type I Interferon ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Type I ◽

Type I Ifn ◽

Activating Mutations ◽

Type I Ifns

Abstract Abstract 3066 Poster Board III-3 Recently, we and others reported activating mutations in JAK1 in acute lymphoblastic leukemia (ALL). These mutations are relatively common in adult patients with T cell ALL. JAK1 is a tyrosine kinase that associates to different cytokine receptors to mediate signal transduction. The associations of the mutant JAK1 with receptors like IL-2R or IL-9R are necessary to promote tumorigenicity by inducing constitutive signaling via the activation of the receptor complex. Because JAK1 mutations confer poor prognosis to the patients, there is a need for new therapies that could specifically target the leukemic blast. Starting from patient samples, we show here that JAK1-mutant ALL blasts are characterized by a type-I interferon (IFN) transcriptional signature. This signature was recapitulated in vitro by the expression of JAK1 mutants in BW5147 and BaF3 hematopoietic cell lines. Binding of JAK1 to the IFN receptor was essential since mutations in the FERM domain abrogated this effect. Beside the constitutive activation of the type I IFN signaling cascade, JAK1 mutations also strongly potentiated the response to IFN in vitro. Typically, the proliferation of cell lines expressing JAK1A634D was abrogated by type I IFNs. Interestingly, we found that different JAK1 mutations differentially potentiate responses to type I IFNs or to IL-9, another cytokine using JAK1 to mediate its effects. This suggests that the type of mutation influences the specificity of the effect on distinct cytokine receptor signaling. Finally, we also showed in an in vivo leukemia model that cells expressing JAK1A634D are hypersensitive to the anti-proliferative and anti-tumorigenic effect of type I IFN, suggesting that type I IFNs should be considered as a potential therapy for ALL with JAK1 activating mutations. Disclosures No relevant conflicts of interest to declare.

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Overcoming Drug Resistance In Acute Lymphoblastic Leukemia by Inhibition of CBP/γ-Catenin

Blood ◽

10.1182/blood.v116.21.3264.3264 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3264-3264

Author(s):

Enzi Jiang ◽

Eugene Park ◽

Cu Nguyen ◽

James Yoon ◽

Yao-Te Hsieh ◽

...

Keyword(s):

Colon Cancer ◽

Western Blot ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Philadelphia Chromosome ◽

Saline Control ◽

Control Group ◽

Apoptosis Protein

Abstract Abstract 3264 Survivin, an inhibitor of apoptosis protein (IAP) family, has been associated with poor prognosis in cancer including leukemia. Survivin can be downregulated in colon cancer cells by inhibition of the β-catenin/Creb-binding protein (CBP) interaction using ICG-001, a small molecule specific inhibitor of the β-catenin/CBP interaction. We have shown previously that combined ICG-001 and chemotherapy can downregulate Survivin and sensitize ALL cells to chemotherapy in vitro and in a pilot study in vivo. In this study, we determine the CBP interaction with ICG-001 in primary ALL cells and preclinically evaluate ICG-001 in vitro and in vivo as an adjuvant against primary ALL and. For this purpose, primary ALL cells were co-cultured with OP9 cells and treated for 4 days with ICG-001 (10mM, 20mM) or DMSO as vehicle control. Mean viability (trypan blue exclusion) of cells treated with ICG-001 was significantly lower (ICG-001 10mM: 75.12% ± 3.15%; 20mM: 41.18%± 7.88%) compared to cells treated with DMSO (84.99% ± 0.42%) (% cell viability relative to initial control) (p=0.03). Real time RT-PCR showed ICG-001 dose-dependent downregulation of Survivin in ALL compared to control (ICG10mM vs. control: p=0.0037 and 20mM vs. control: p=0.0031). Immunoblotting demonstrated reduction of Survivin after ICG-001 treatment. Primary ALL cells incubated with a combination of VDL (Vincristine, Dexamethasone and L-Asparaginase) and ICG-001 showed decreased viability (28.7%± 4.9%) versus VDL only (79.3%± 13.6%) (p=0.014) determined by MTT assay. To elucidate if ICG-001 interacts with β-catenin/CBP as shown previously in colon cancer, we analyzed ten primary pre-B ALL cells and found significantly greater γ-catenin and Survivin expression versus normal pre-B-Cells. β-catenin was absent or in some cases expressed only weakly. Expression of v-catenin and b-catenin in ALL xenograft cells were detected by Western blot. One primary ALL was selected and incubated with γ-catenin and β-catenin siRNA for 48hrs, followed by 6hrs incubation with Wnt3a. Wnt3a induced both of γ-catenin and β-catenin expression. Survivin was reduced by γ-catenin siRNA but not β-catenin siRNA treatment. Addition of Wnt3a partially recovered the decrease of Survivin. In addition, Survivin was knocked down in primary ALL using shRNA and non-silencing shRNA control or ICG-001 (2uM) and DMSO control. Western blot analysis showed that survivin shRNA or ICG-001 treatment lead to downregulation of Survivin and γ-catenin. Using a ChIP assay we could demonstrate occupancy of TCF4 and CBP association at the Survivin promoter, which was not altered by ICG-001 in primary ALL. Moreover, ICG-001 treatment of primary ALL cells prevents CBP but not p300 occupancy. For further preclinical in vivo evaluation of ICG-001, one Philadelphia chromosome positive ALLs (Ph+) and two Ph− primary ALL were injected into sublethally irradiated NOD/SCID IL2Rγ−/-mice and treated with ICG-001 (50mg or 100mg/kg/day per subcutaneous miniosmotic pump) with or without chemotherapy including VDL for Ph− ALL (per intraperitoneal injections) or Nilotinib for Ph+ ALL (per os). For analysis we pooled the survival of all three primary leukemias. The saline control group (n=10) (MST= 55.5.days) and the ICG-001 only groups (n=3) (MST=61 days) died rapidly. The group treated with chemotherapy (n=13) had a median survival time (MST) of 85 days. In marked contrast, the group treated with the combined chemotherapy+ICG-001 (n=15) lived significantly longer (MST=100) (p<0.05). Taken together, our data shows that Survivin transcription can be mediated by γ-catenin in primary ALL and that targeting CBP/γ-catenin by using ICG-001 ALL can sensitize ALL cells to chemotherapy in vitro and in vivo. Disclosures: No relevant conflicts of interest to declare.

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The Natural Flavonoid Naringenin Inhibits the Cell Growth of WilmsTumorinChildren by Suppressing TLR4/NF-κB Signaling

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620999200818155814 ◽

2020 ◽

Vol 20 ◽

Author(s):

Hongtao Li ◽

Peng Chen ◽

Lei Chen ◽

Xinning Wang

Keyword(s):

Cell Growth ◽

Western Blot ◽

Wilms Tumor ◽

Critical Role ◽

Time Dependent ◽

Luciferase Assay ◽

Dependent Manner ◽

Tlr4 Expression ◽

Protein Levels

Background: Nuclear factor kappa B (NF-κB) is usually activated in Wilms tumor (WT) cells and plays a critical role in WT development. Objective: The study purpose was to screen a NF-κB inhibitor from natural product library and explore its effects on WT development. Methods: Luciferase assay was employed to assess the effects of natural chemical son NF-κB activity. CCK-8 assay was conducted to assess cell growth in response to naringenin. WT xenograft model was established to analyze the effect of naringenin in vivo. Quantitative real-time PCR and Western blot were performed to examine the mRNA and protein levels of relative genes, respectively. Results: Naringenin displayed significant inhibitory effect on NF-κB activation in SK-NEP-1 cells. In SK-NEP-1 and G-401 cells, naringenin inhibited p65 phosphorylation. Moreover, naringenin suppressed TNF-α-induced p65 phosphorylation in WT cells. Naringenin inhibited TLR4 expression at both mRNA and protein levels in WT cells. CCK-8 staining showed that naringenin inhibited cell growth of the two above WT cells in dose-and time-dependent manner, whereas Toll-like receptor 4 (TLR4) over expression partially reversed the above phenomena. Besides, naringenin suppressed WT tumor growth in dose-and time-dependent manner in vivo. Western blot found that naringenin inhibited TLR4 expression and p65 phosphorylation in WT xenograft tumors. Conclusion: Naringenin inhibits WT development viasuppressing TLR4/NF-κB signaling

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TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽

10.1186/s12885-021-08562-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Chengwu Xiao ◽

Wei Zhang ◽

Meimian Hua ◽

Huan Chen ◽

Bin Yang ◽

...

Keyword(s):

Cell Lines ◽

Prognostic Indicator ◽

The Cancer Genome Atlas ◽

Mrna Levels ◽

Loss Of Function ◽

Protein Levels ◽

Kaplan Meier ◽

Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

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Novel CD19/CD22 Bicistronic Chimeric Antigen Receptors Outperform Single or Bivalent Cars in Eradicating CD19+CD22+, CD19-, and CD22- Pre-B Leukemia

Blood ◽

10.1182/blood.v130.suppl_1.810.810 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 810-810 ◽

Cited By ~ 3

Author(s):

Haiying Qin ◽

Sang M Nguyen ◽

Sneha Ramakrishna ◽

Samiksha Tarun ◽

Lila Yang ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Single Chain ◽

Dual Targeting ◽

Single Antigen ◽

Therapeutic Function

Abstract Treatment of pre-B cell acute lymphoblastic leukemia (ALL) using chimeric antigen receptor expressing T cells (CART) targeting CD19 have demonstrated impressive clinical results in children and young adults with up to 70-90% complete remission rate in multiple clinical trials. However, about 30% of patients relapse due to loss of the targeted epitope on CD19 or CART failure. Our CD22-targeted CAR trial has generated promising results in relapsed/refractory ALL, including CD19 antigen negative ALL, but relapse associated with decreased CD22 site density has occurred. Thus, developing strategies to prevent relapses due to changes in antigen expression have the potential to increase the likelihood of durable remissions. In addition, dual targeting of both CD19 and CD22 on pre-B ALL may be synergistic compared to targeting a single antigen, a potential approach to improve efficacy in patients with heterogeneous expression of CD19 and CD22 on leukemic blasts. We describe the systematic development and comparison of the structure and therapeutic function of three different types (over 15 different constructs) of novel CARs targeting both CD19 and CD22: (1) Bivalent Tandem CAR, (2) Bivalent Loop CAR, and (3) Bicistronic CAR. These dual CARs were assembled using CD19- and CD22-binding single chain fragment variable (scFv) regions derived from clinically validated single antigen targeted CARs. They are structurally different in design: both tandem and loop CARs have the CD19 and CD22 scFv covalently linked in the same CAR in different orders, whereas, bicistronic CARs have 2 complete CAR constructs connected with a cleavable linker. The surface expression on the transduced T cell of the CD19/CD22 dual CARs was detected with CD22 Fc and anti-idiotype of CD19 and compared to single CD19 or CD22 CARs. Activities of dual CARs to either CD19 or CD22 were evaluated in vitro with cytotoxicity assays or killing assays against K562 cells expressing either CD19 or CD22 or both antigens and also tested against a leukemia CD19+/CD22+ cell line, NALM6, and NALM6 with CRISPER/CAS9 knockout of CD19 or CD22 or both antigens. Therapeutic function of the top candidates of the dual CARs was then validated in vivo against these NALM6 leukemia lines. Some of these dual CARs were also further tested against patient-derived xenografts. Finally, we tested the dual targeting CARs in an artificial relapse model in which mice were co-injected with a mix of CD19 knockout and CD22 knockout NALM6 leukemia lines. From these studies, we established that the order of the scFv, size of the linker, type of leader sequence, and co-stimulatory domain in the CAR constructs all impact the efficacy of the dual targeting CARs. Tandem, Loop, and Bicistronic CARs all demonstrate some levels of in vitro and in vivo activities, but the bicistronic CAR was most effective at clearing leukemia and preventing relapse. In the CD19+/CD22+ NALM6 model, bicistronic CAR treated mice remain disease free while CD19 CAR or CD22 CAR treated mice already died or relapsed on day 27. In the relapse model, as expected, CD19 or CD22 single CAR T cell treatment resulted in progression of the corresponding antigen-negative NALM6. Treatment with dual targeted bicistronic CARs resulted in clearance of both CD19 and CD22 negative ALL with durable remission. In summary, we described novel CD19/CD22 dual targeting CARs with robust pre-clinical activity against pre-B cell ALL, and validated this approach in the prevention of resistance to single-antigen targeted CARs in preclinical models. Disclosures No relevant conflicts of interest to declare.

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Identification of Prostaglandin F2 Receptor Negative Regulator (PTGFRN) as an internalizable target in cancer cells for antibody-drug conjugate development

PLoS ONE ◽

10.1371/journal.pone.0246197 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0246197

Author(s):

Jorge Marquez ◽

Jianping Dong ◽

Chun Dong ◽

Changsheng Tian ◽

Ginette Serrero

Keyword(s):

Flow Cytometry ◽

Western Blot ◽

Cell Lines ◽

Cancer Cell ◽

Cancer Cell Lines ◽

Negative Regulator ◽

Target Validation ◽

Antibody Drug

Antibody-drug conjugates (ADC) are effective antibody-based therapeutics for hematopoietic and lymphoid tumors. However, there is need to identify new targets for ADCs, particularly for solid tumors and cancers with unmet needs. From a hybridoma library developed against cancer cells, we selected the mouse monoclonal antibody 33B7, which was able to bind to, and internalize, cancer cell lines. This antibody was used for identification of the target by immunoprecipitation and mass spectrometric analysis, followed by target validation. After target validation, 33B7 binding and target positivity were tested by flow cytometry and western blot analysis in several cancer cell lines. The ability of 33B7 conjugated to saporin to inhibit in vitro proliferation of PTFRN positive cell lines was investigated, as well as the 33B7 ADC in vivo effect on tumor growth in athymic mice. All flow cytometry and in vitro internalization assays were analyzed for statistical significance using a Welsh’s T-test. Animal studies were analyzed using Two-Way Analysis of Variance (ANOVA) utilizing post-hoc Bonferroni analysis, and/or Mixed Effects analysis. The 33B7 cell surface target was identified as Prostaglandin F2 Receptor Negative Regulator (PTGFRN), a transmembrane protein in the Tetraspanin family. This target was confirmed by showing that PTGFRN-expressing cells bound and internalized 33B7, compared to PTGFRN negative cells. Cells able to bind 33B7 were PTGFRN-positive by Western blot analysis. In vitro treatment PTGFRN-positive cancer cell lines with the 33B7-saporin ADC inhibited their proliferation in a dose-dependent fashion. 33B7 conjugated to saporin was also able to block tumor growth in vivo in mouse xenografts when compared to a control ADC. These findings show that screening antibody libraries for internalizing antibodies in cancer cell lines is a good approach to identify new cancer targets for ADC development. These results suggest PTGFRN is a possible therapeutic target via antibody-based approach for certain cancers.

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Irinotecan Resistance – In Search of New Markers in CRC.

10.21203/rs.3.rs-1088696/v1 ◽

2021 ◽

Author(s):

Jakub Kryczka ◽

Joanna Boncela

Keyword(s):

Drug Resistance ◽

Cell Lines ◽

Expression Profiles ◽

Resistance Mechanism ◽

Interaction Network ◽

In Vitro Study ◽

Cancer Drug ◽

New Genes

Abstract Colorectal cancer (CRC) is one of the most prominent causes of cancer death worldwide. Chemotherapeutic regimens consisting of different drugs combinations such as 5-fluorouracil, and oxaliplatin (FOLFOX) or irinotecan (FOLFIRI) have been proven successful in the treatment of CRC. However, chemotherapy often leads to the acquisition of cancer drug resistance followed by metastasis and in the aftermath therapeutic failure. The molecular mechanism responsible for drug resistance is still unclear. The systemic search for new biomarkers of this phenomenon may identify new genes and pathways. To understand the drug resistance mechanism in CRC, the in vitro study based on the molecular analysis of drug-sensitive cells lines vs drug-resistant cells lines has been used. In our study to bridge the gap between in vitro and in vivo study, we compared the expression profiles of cell lines and patient samples from the publicly available database to select the new candidate genes for irinotecan resistance. Using The Gene Expression Omnibus (GEO) database of CRC cell lines (HT29, HTC116, LoVo, and their respective irinotecan-resistant variants) and patient samples (GSE42387, GSE62080, and GSE18105) we compared the changes in the mRNA expression profile of the main genes involved in irinotecan body’s processing, such as transport out of the cells and metabolism. Furthermore, using a protein-protein interaction network of differently expressed genes between FOLFIRI resistant and sensitive CRC patients, we have selected top networking proteins (upregulated: NDUFA2, SDHD, LSM5, DCAF4, and COX10, downregulated: RBM8A, TIMP1, QKI, TGOLN2, and PTGS2). Our analysis provided several potential irinotecan resistance markers, previously not described as such.

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Exosomal miR-25-3p from mesenchymal stem cells alleviates myocardial infarction by targeting pro-apoptotic proteins and EZH2

Cell Death and Disease ◽

10.1038/s41419-020-2545-6 ◽

2020 ◽

Vol 11 (5) ◽

Cited By ~ 1

Author(s):

Yi Peng ◽

Ji-Ling Zhao ◽

Zhi-Yong Peng ◽

Wei-Fang Xu ◽

Guo-Long Yu

Keyword(s):

Myocardial Infarction ◽

Inflammatory Responses ◽

Luciferase Assay ◽

Protective Effects ◽

Functional Studies ◽

Protein Levels ◽

Cardioprotective Effects ◽

Apoptotic Proteins

Abstract Mesenchymal stem cell (MSC) therapy is a promising approach against myocardial infarction (MI). Studies have demonstrated that MSCs can communicate with other cells by secreting exosomes. In the present study, we aimed to identify exosomal microRNAs that might contribute to MSC-mediated cardioprotective effects. Primary cardiomyocytes were deprived of oxygen and glucose to mimic MI in vitro. For the animal model of MI, the left anterior descending artery was ligated for 1 h, followed by reperfusion for 12 h. MSC-derived exosomes were used to treat primary cardiomyocytes or mice. Cardioprotection-related microRNAs were determined, followed by target gene identification and functional studies with quantitative PCR, western blotting, MTT assay, flow cytometry assay, chromatin immunoprecipitation and dual-luciferase assay. We found that MSC co-culture reduced OGD-induced cardiomyocyte apoptosis and inflammatory responses. Cardioprotection was also observed upon treatment with MSC-derived exosomes in vitro and in vivo. In line with this, exosome uptake led to a significant increase in miR-25-3p in cardiomyocytes. Depletion of miR-25-3p in MSCs abolished the protective effects of exosomes. Mechanistically, miR-25-3p directly targeted the pro-apoptotic genes FASL and PTEN and reduced their protein levels. Moreover, miR-25-3p decreased the levels of EZH2 and H3K27me3, leading to derepression of the cardioprotective gene eNOS as well as the anti-inflammatory gene SOCS3. Inhibition of EZH2 or overexpression of miR-25-3p in cardiomyocytes was sufficient to confer cardioprotective effects in vitro and in vivo. We concluded that exosomal miR-25-3p from MSCs alleviated MI by targeting pro-apoptotic proteins and EZH2.

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Colorectal Cancer Growth Retardation through Induction of Apoptosis, Using an Optimized Synergistic Cocktail of Axitinib, Erlotinib, and Dasatinib

Cancers ◽

10.3390/cancers11121878 ◽

2019 ◽

Vol 11 (12) ◽

pp. 1878 ◽

Cited By ~ 6

Author(s):

Robert H. Berndsen ◽

Nathalie Swier ◽

Judy R. van Beijnum ◽

Patrycja Nowak-Sliwinska

Keyword(s):

Colorectal Cancer ◽

Cell Lines ◽

Kinase Inhibitors ◽

Expression Profiles ◽

Chorioallantoic Membrane ◽

Treatment Strategies ◽

Tumor Growth Inhibition ◽

Clinical Implementation

Patients with advanced colorectal cancer (CRC) still depend on chemotherapy regimens that are associated with significant limitations, including resistance and toxicity. The contribution of tyrosine kinase inhibitors (TKIs) to the prolongation of survival in these patients is limited, hampering clinical implementation. It is suggested that an optimal combination of appropriate TKIs can outperform treatment strategies that contain chemotherapy. We have previously identified a strongly synergistic drug combination (SDC), consisting of axitinib, erlotinib, and dasatinib that is active in renal cell carcinoma cells. In this study, we investigated the activity of this SDC in different CRC cell lines (SW620, HT29, and DLD-1) in more detail. SDC treatment significantly and synergistically decreased cell metabolic activity and induced apoptosis. The translation of the in-vitro-based results to in vivo conditions revealed significant CRC tumor growth inhibition, as evaluated in the chicken chorioallantoic membrane (CAM) model. Phosphoproteomics analysis of the tested cell lines revealed expression profiles that explained the observed activity. In conclusion, we demonstrate promising activity of an optimized mixture of axitinib, erlotinib, and dasatinib in CRC cells, and suggest further translational development of this drug mixture.

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