Mcl-1 mRNA Levels Research Articles

Knockdown of DNMT1 Induces SLCO3A1 to Promote Follicular Growth by Enhancing the Proliferation of Granulosa Cells in Mammals.

In female mammals, the proliferation and apoptosis of granulosa cells (GCs) have been shown to determine the fate of follicles. DNA methyltransferases (DNMTs) and SLCO3A1 have been reported to be involved in the survival of GCs and follicular growth. However, the molecular mechanisms enabling DNMTs to regulate the expression of SLCO3A1 to participate in follicular growth are unclear. In this study, we found that the knockdown of DNMT1 enhanced the mRNA and protein levels of SLCO3A1 by regulating the chromatin accessibility probably. Moreover, SLCO3A1 upregulated the mRNA and protein levels of MCL1, PCNA, and STAR to promote the proliferation of GCs and facilitated cell cycle progression by increasing the mRNA and protein levels of CCNE1, CDK2, and CCND1, but it decreased apoptosis by downregulating the mRNA and protein levels of CASP3 and CASP8. Moreover, SLCO3A1 promoted the growth of porcine follicles and development of mice follicles. In conclusion, the knockdown of DNMT1 upregulated the mRNA and protein levels of SLCO3A1, thereby promoting the proliferation of GCs to facilitate the growth and development of ovarian follicles, and these results provide new insights into investigations of female reproductive diseases.

Formononetin and Dihydroartemisinin Act Synergistically to Induce Apoptosis in Human Acute Myeloid Leukemia Cell Lines.

Enhanced cell survival and drug resistance in tumor cells have been linked to the overexpression of antiapoptotic members of the Bcl-2 family proteins, including Bcl-2 and Mcl-1. The aim of this study was to explore the impact of formononetin and dihydroartemisinin combination on the growth and apoptosis of acute myeloid leukemia (AML) cells. In this experimental study, the cell survival and cell proliferation were tested by MTT assay and trypan blue staining. The evaluation of cell apoptosis was conducted using Hoechst 33342 staining and a colorimetric assay to measure caspase-3 activity. To determine the mRNA levels of Mcl-1, Bcl-2, Bax, and Cyclin D1, a quantitative real-time polymerase chain reaction (qRT-PCR) was performed. We showed that treatment with either formononetin or dihydroartemisinin alone, led to significant decrease in the cell survival and growth, and triggered apoptosis in U937 and KG-1 AML cell lines. Moreover, treatment with each of the compounds alone significantly decreased the mRNA levels of Mcl-1, Bcl-2 and Cyclin D1 mRNA, while, the expression level of Bax mRNA was enhanced. Combination of two compounds showed a synergistic anti-cancer effect. The anti-leukemic potential of formononetin and dihydroartemisinin is exerted through the effect on cell cycle progression and intrinsic pathway of apoptosis. Therefore, they can be considered as a potential anti-leukemic agent alone or along with existing chemotherapeutic drugs.

The Effects of ABT-199 and Dihydroartemisinin Combination on Cell Growth and Apoptosis in Human U937 and KG-1 Cancer Cells.

Change in the balance of Bcl-2 family proteins is one of the main reasons for resistance of tumor cells to ABT-199. In this study, the effect of dihydroartemisinin on cell growth, apoptosis and sensitivity of the AML cells to ABT-199 was investigated. Cell proliferation and survival were assessed by trypan blue staining and MTT assay, respectively. Cell apoptosis was measured by Hoechst 33342 staining and caspase-3 activity assay. The expression levels of Bcl-2, Mcl-1 and Bax mRNA were tested by qRT-PCR. Our data showed that combination therapy significantly reduced the IC50 value and synergistically decreased the AML cell survival and growth compared with dihydroartemisinin or ABT-199 alone. Treatment with each of ABT-199 or dihydroartemisinin alone clearly enhanced the Bax mRNA expression and inhibited the expression of Mcl-1 and Bcl-2 mRNA. Inhibition of Mcl-1 mRNA by dihydroartemisinin was associated with enhancement of apoptosis induced by ABT-199 in AML cells. In conclusion, dihydroartemisinin not only triggers the intrinsic pathway of apoptosis, but also can increase the sensitivity of the AML cells to ABT-199 via suppression of Mcl-1 expression.

Dihydroartemisinin Enhances the Therapeutic Efficacy of BH3 Mimetic Inhibitor in Acute Lymphoblastic Leukemia Cells via Inhibition of Mcl-1.

Up-regulation of the anti-apoptotic proteins such as Mcl-1 is associated with the primary and secondary resistance of tumor cells to ABT-737 Bcl-2 inhibitor. The combined treatment of Bcl-2 inhibitors with Mcl-1 inhibitors has been proposed as an attractive therapeutic strategy to overcome this drug resistance. Here, we investigated the effect of dihydroartemisinin on Mcl-1 expression and sensitization of T-ALL cells to ABT-737. The cell growth and survival were tested by the cell proliferation and MTT assays, respectively. The mRNA levels of Bcl-2, Mcl-1, Bax and P21 were examined by qRT-PCR. Apoptosis were detected by Hoechst 33342 staining and caspase-3 activity assay. Our data showed that combination treatment with dihydroartemisinin and ABT-737 caused a significant decrease in the IC50 value and synergistically reduced the cell survival compared with dihydroartemisinin or ABT-737 alone. ABT-737 enhanced the Mcl-1 mRNA expression. Dihydroartemisinin also down-regulated the expression of Bcl-2 and Mcl-1 and enhanced the P21 and Bax expression. Moreover, dihydroartemisinin enhanced the apoptosis induced by ABT-737 in MOLT-4 and MOLT-17 cell lines. In conclusion, dihydroartemisinin demonstrates anti-tumor activities in human ALL cells via inhibition of cell survival and growth. Dihydroartemisinin augments the apoptotic effect of ABT-737 by inhibiting the expression of Mcl-1.

Exploring the Potential of Isalo Scorpion Cytotoxic Peptide in Enhancing Gill Barrier Function and Immunity in Grass Carp (Ctenopharyngodon idella) Infected with Flavobacterium columnare

The objective of this research was to investigate how dietary antimicrobial peptides (AMP), namely, Isalo scorpion cytotoxic peptide (IsCT), affect the gill physical barrier function and immune function of grass carp challenged with Flavobacterium columnare (F. columnare). Five hundred forty grass carp were randomly allocated to six groups and fed to varying levels of IsCT in the diet (0, 0.6, 1.2, 1.8, 2.4, and 3.0 mg/kg diet) for a duration of 60 days. Afterward, the grass carps faced a challenge from F. columnare. The results revealed that the use of optimal IsCT dramatically mitigated gill damage in grass carp that were infected with F. columnare. Additionally, IsCT exhibited a notable enhancement in gill antioxidant capabilities, as evidenced by a significant reduction in ROS, MDA, and PC levels, an elevation in antioxidant enzyme activities, and an upregulation of antioxidant‐related genes and Nrf2 mRNA levels. Conversely, the expression of Keap1a and Keap1b mRNA was decreased. Besides, IsCT exhibited its capability to inhibit apoptosis via downregulating the mRNA levels of caspase-2, caspase-3, caspase-7, caspase-8, caspase-9, Apaf1, Fasl, Bax, and JNK while concurrently increasing the mRNA levels of Bcl-2, Mcl-1, and IAP in fish gills. Additionally, IsCT promoted the integrity of tight junction barrier by increasing the gene expression of claudin-b, claudin-c, claudin-3c, ZO-1, ZO-2b, occludin, and JAM while suppressing MLCK signaling. Additionally, optimal dietary IsCT improved antibacterial ability, as evidenced by heightened LZ, ACP activities, and elevated levels of C3, C4, and IgM. Additionally, there was an upregulation in β-defensin-1, LEAP-2A, LEAP-2B, hepcidin, and mucin-2 mRNA expression in the gills. Simultaneously, the inclusion of optimal dietary IsCT in the diet resulted in improved gill immunity barriers through the reduction of proinflammatory cytokine mRNA levels and the increase in the expression of anti‐inflammatory cytokine mRNA levels. This was partly facilitated by the IκBa/NF‐κB p65 signaling pathway and TOR/S6K1 signaling pathways in the gills of grass carp. Therefore, supplementing the diet with IsCT has potential advantages in enhancing gill health by improved physical barriers and immunity in grass carp. Based on LZ activity and against lipid peroxidation, optimum IsCT concentrations in on‐growing grass carp (136.88 ± 0.72 g) were found to be 1.68 and 1.54 mg/kg diet, respectively.

CDK9 Inhibition Overcomes Ibrutinib Resistance in Mantle Cell Lymphoma (MCL)

Introduction: Bruton tyrosine kinase (BTK) inhibitors (e.g., ibrutinib [ibr]) have shown clinical efficacy in MCL. However, resistance to BTK inhibitors is inevitable. Cyclin-dependent kinases (CDKs)are Ser/Thr kinases that associate with specific cyclins during the cell cycle to regulate its progression. We and others have shown that selective targeting of CDK9, a pTEFb component which facilitates RNA transcription, downmodulates MYC and MCL1 in malignant B-cells. Here, we investigated the effects of a selective CDK9 inhibitor, AZD4573, in ibr-naive and ibr-resistant MCL in vitro and in vivo. Methods: MCL cell lines (Mino, JeKo-1) were cultured in increasing doses of ibr for ~6 months to generate resistance. MCL cell lines were treated with AZD4573 and assayed for apoptosis/proliferation, metabolic phenotype (Seahorse), and protein and RNA expression. Primary MCL cells were incubated for 24 h in CD40L/BAFF-conditioned media prior to drug treatment. Two MCL PDX models were used (chemo-resistant and ibr-resistant). MCL cells were injected IV in NSG mice and tracked by flow cytometry (CD5 + CD19 + CD45 +). Upon MCL detection in the peripheral blood (~1-2%), mice began treatment with AZD4573 (25 mg/kg IP once or twice weekly) or vehicle control. Splenocytes were harvested and RNA was isolated and analyzed by RNA-Seq/RT-PCR. Additionally, primary circulating MCL cells were collected from a patient treated with one dose of AZD4573 (at baseline; 4 and 7 h post-infusion and day 7) and analyzed by scRNA-Seq. Results: Treatment with AZD4573 potently inhibited proliferation and induced apoptosis of both ibr-sensitive and resistant MCL cell lines in vitro (IC 50 ~ 2-15 nM). AZD4573 also induced apoptosis of primary MCL cells in stromal conditions. Loss of RNA polymerase phosphorylation at Ser2 residue, a CDK9-dependent site, preceded apoptosis as measured by cleaved PARP. AZD4573 did not inhibit CDKs 2, 4/6, and 7 (as measured by pRb and pRNApol [Ser5]), confirming its selective effect. CDK9 inhibition resulted in rapid and dose-dependent downregulation of MYC and MCL1 mRNA and protein levels in MCL cell lines and primary cells. Treatment with AZD4573 but not with ibr extended survival of ibr-resistant PDX mice without significant toxic events. RNA-Seq analysis of the MCL splenocytes harvested from these mice after 3 weeks of AZD4573 treatment demonstrated that gene sets related to transcription-factor activity were most enriched in the treated samples, consistent with known drug mechanism. TNFa/NFκB, TGF-β and PI3K/AKT/mTOR signaling pathways were most downregulated. We have shown that CDK9 inhibition leads to epigenetic reprogramming in neoplastic B-cells, resulting in upregulation of a subset of superenhancer-associated oncogenes, i.e. MYC, PIM kinaseand others (Thieme et al, 2023). Consistent with this notion, splenocytes derived from AZD4573-treated mice exhibited upregulation of MYC-dependent targets and OxPhos at the time of sacrifice. A number of genes relating to components of the electron transport chain were upregulated ( ATP5F1D, NDUFS7, ATP5MC1 as the top three). This suggested that these pathways may govern resistance to CDK9 inhibition in MCL over time. To further explore this, parental and ibr-resistant MCL cell lines were treated with low doses of AZD4573 (~2 nM). After 48 h, CDK9 inhibition was associated with increased oxygen consumption rates (OCR) in Seahorse analysis. This was not accompanied by increased cell proliferation by cell cycle analysis or Ki-67 staining. IACS-010759, an inhibitor of complex I of the ETC which targets OxPhos, demonstrated synergy with AZD4573 in parental and ibr-resistant MCL cell lines. Single-cell RNA-Seq analysis of primary MCL cells obtained from a patient treated with AZD4573 on a clinical trial revealed rapid downmodulation of NFκB signaling (at 4 h) and MYC transcriptional targets and PI3K/AKT/mTOR signaling pathways (at 7 h). Interestingly, we observed partial recovery of MYC, mTOR pathways and induction of OxPhos by day 7 (prior to next planned dose), validating our pre-clinical findings. Conclusion: CDK9 inhibition with AZD4573 induced apoptosis, downregulated MYC and MCL1 and NFκB signaling, and overcame ibr resistance in preclinical MCL models. This was also noted in a sample obtained from a clinical trial. Prolonged CDK9 inhibition led to metabolic reprogramming towards OxPhos, which thus can serve as a therapeutic target in MCL.

AL amyloidosis clonal plasma cells are regulated by microRNAs and dependent on anti-apoptotic BCL2 family members.

Noncoding RNAs such as microRNAs (miRNAs) have attracted attention as biological pathway regulators, which differ from chromosomal translocations and gene point mutations. Their involvement in the molecular mechanisms underlying light chain (AL) amyloidosis pathogenesis is yet to be elucidated. To decipher specific miRNA expression profile in AL-amyloidosis and to examine how miRNAs are involved in AL pathogenesis. The expression profile of miRNAs and mRNA from bone marrow (BM)-derived CD138+ cells were determined using the NanoString nCounter assay and RNA-Seq, respectively. The effect of aberrantly expressed miRNAs on potential molecular targets was analyzed by qRT-PCR, Western blot, Mito-potential assay, and Annexin-PI staining. Genes which were significantly differentially expressed between AL-amyloidosis and MM, were found to be involved in cell growth and apoptotic mechanisms. Specifically, BCL2L1, MCL1, and BCL2 were upregulated in AL-amyloidosis compared with MM and controls. The levels of miR-181a-5p and miR-9-5p, which regulate the above-mentioned genes, were lower in BM samples from AL-amyloidosis compared with controls, providing a mechanism for BCL2 family gene upregulation. When miR-9-5p and miR-181a-5p were overexpressed in ALMC1 cells, BCL2L1, MCL1, and BCL2 were downregulated and induced apoptosis. Treatment of ALMC-1 cells with venetoclax, (BCL-2 inhibitor), resulted in the upregulation of those miRNAs, the downregulation of BCL2, MCL1, and BCL2L1 mRNA and protein levels, and subsequent apoptosis. Our findings suggest that miR-9-5p and miR-181a-5p act as tumor-suppressors whose downregulation induces anti-apoptotic mechanisms underlying the pathogenesis of AL-amyloidosis. The study highlights the post-transcriptional regulation in AL-amyloidosis and provides pathogenetic evidence for the potential use of BCL-2 inhibitors in this disease.

FOXO1 represses MCL1 transcription to regulate the function of vascular smooth muscle cells in intracranial aneurysm.

Intracranial aneurysm (IA) is a pathological dilation of the cerebral arteries. Vascular smooth muscle cell (VSMC) dysfunction assumes a role in IA development. In this context, this study probed the role of FOXO1 in human brain VSMC (HBVSMC) function via MCL1. FOXO1 and MCL1 expression in arterial wall tissues from IA patients and inflammatory cytokines (IL-1β, TNF-α, and IL-6) levels in the serum of IA patients were, respectively, detected with qRT-PCR and ELISA. Pearson's correlation analysis was utilized to analyze the correlation between FOXO1 and MCL1. After FOXO1 and/or MCL1 were overexpressed in HBVSMCs, caspase-3 and Cyt-c protein expression were examined by western blot, cell proliferation by CCK-8 and EdU assays, and cell apoptosis by flow cytometry. IL-1β, TNF-α, and IL-6 levels were assessed in the supernatant of HBVSMCs with ELISA. Dual-luciferase gene reporter and ChIP assays were conducted to evaluate the binding of FOXO1 to MCL1. FOXO1 expression was high and MCL expression was low in arterial wall tissues from IA patients, and IL-1β, TNF-α, and IL-6 levels were high in the serum of IA patients. There was an inverse correlation between FOXO1 and MCL1 mRNA levels. Moreover, FOXO1 bound to the MCL1 promoter to decrease MCL1 transcription. In addition, FOXO1 overexpression augmented cell apoptosis, caspase-3 and Cyt-c protein expression, and IL-1β, TNF-α, and IL-6 secretion, while reducing cell proliferation in HBVSMCs, which was abrogated by further MCL1 overexpression. FOXO1 impeded MCL1 transcription to curb HBVSMC proliferation and facilitate their apoptosis and inflammation.

Dual inhibition of BCL2L1 and MCL1 is highly effective against RET fusion-positive or MET exon 14 skipping mutation-positive lung adenocarcinoma cells

Non-small cell lung carcinomas (NSCLCs), especially lung adenocarcinomas (LUADs), harbor several driver mutations against which highly effective tyrosine kinase inhibitors (TKIs) are available. Although TKIs are generally effective against certain NSCLCs, primary or acquired resistance almost always develops. Driver mutations include RET fusion (∼1–2% of NSCLC cases) and MET exon 14 skipping mutation (METΔex14; ∼3–4%). Surprisingly, the LUAD cell line LC-2/ad with CCDC6-RET fusion thrived independently of RET signaling, and Hs-746T cells harboring METΔex14 plus amplification survived MET silencing. However, these two cell lines were highly sensitive to dual silencing of the representative anti-apoptotic BCL2 family members BCL2L1 and MCL1, undergoing extensive apoptosis in monolayer or 3D on-top culture systems. Moreover, we found that most LUAD cell lines and tissues expressed high levels of BCL2L1 and MCL1 mRNA but extremely low levels of BCL2. Together, these findings suggest that inhibiting BCL2L1 plus MCL1 may represent a new approach to treating LUAD cells irrespective of their driver mutations.

LS-007 inhibits melanoma growth via inducing apoptosis and cell cycle arrest and regulating macrophage polarization.

LS-007, an inhibitor of cyclin-dependent kinase 9 (CDK9), exhibits potential antitumor activity against chronic lymphocytic leukemia and ovarian cancer, but its effect on melanoma and tumor microenvironment (TME) has not been reported yet. This study aimed to investigate the role of LS-007 in B16F10 melanoma and relevant mechanisms. LS-007 significantly inhibited viability and induced apoptosis of B16F10 cells in a dose-dependent manner, which were accompanied with the increased ratio of Bax to Bcl-2 and decreased Mcl-1 mRNA level. Western blot analysis showed that LS-007 increased the expression of cleaved caspase-3 and poly ADP-ribose polymerase (PARP). Furthermore, flow cytometry analysis and qRT-PCR results showed that LS-007 treatment resulted in cell cycle arrest by changing cell cycle-related gene expression. Notably, in vivo evaluation showed that LS-007 significantly decreased the weight and volume of tumor and the expression of Ki67, promoted the expression of iNOS and inhibited the expression of CD206, suggesting that LS-007 might inhibit tumor growth by suppressing polarization of macrophages into tumor-associated macrophages (TAMs) in the TME. The increase in M1/M2 treated with LS-007 detected by flow cytometry hinted that macrophages were polarized towards an antitumor phenotype. In addition, LS-007 induced higher apoptotic rate of B16F10 cells when co-cultured B16F10 with BMDMs. LS-007 has inhibitory effects on B16F10 cells in vivo and in vitro via inducing apoptosis, cell cycle arrest, and changing macrophage function in the TME.

CD56 Has a Critical Role in Regulating Multiple Myeloma Cell Growth and Response to Therapies

Multiple myeloma (MM) is a disease derived from genetically abnormal clonal plasma cells. MM cells aberrantly express several surface antigens compared with normal plasma cells. Among others, CD56/NCAM1 is present at variable levels in approximately 70% of MM patients. Very little is known about its role in MM; however, CD56 positivity in MM correlates with greater osteolytic burden and lower frequency of good prognostic features, such as the presence of t(11;14).We first analyzed 569 patients with MM diagnosed between 1/1/2005 and 12/31/2014 at the Ohio State University Wexner Medical Center, stratifying them based on the percentage of CD56-expressing clonal MM cells. The mean percentage of CD56-expressing clonal MM was 26.5%, with range from 0 to 100%; the Mean Fluorescent Intensity values varied, with a quarter of patients expressing CD56 at high intensity. We then evaluated patient outcomes based on the percentage of CD56-expressing clonal MM cells. We noticed that MM patients with more than 30 or 50 percent of CD56-expressing MM clonal cells have inferior clinical outcomes than patients with less than 30 or 50 percent of CD56-expressing MM clonal cells, with median overall survival of 9.61 versus 7.64 years (log-rank p-value: 0.004) or 9.30 versus 6.47 years (log-rank p-value: 0.0009), respectively.We then demonstrated by conventional and real-time PCR analyses that the predominately expressed CD56 isoform in MM has signaling potential with a transmembrane portion and cytosolic tail. Therefore, we evaluated the functional role of CD56 in MM. By gain-of function studies in U266 and MM.1S MM cell lines, we showed that overexpression of CD56 promotes MM growth and viability; the opposite effect occurred with CD56 silencing in H929, OPM-2, and RPMI-8226 MM cell lines, which leads to reduced MM growth and increased apoptotic cell death. Overexpression of CD56 resulted in the phosphorylation and hence activation of ribosomal protein S6 kinase A3 (RSK2) and of the transcription factor, cAMP responsive element binding protein 1 (CREB1). This induced CREB1 binding to DNA consensus CRE elements, and promoted transcription of CREB1 targets, the anti-apoptotic genes BCL2 and MCL1. CD56 silencing in H929 and OPM-2 MM cell lines had opposite effects, with reduction of phospho-RSK2, phospho-CREB1, MCL1, and BCL2 levels.We then used shRNAs targeting RSK2 and CREB1 or specific inhibitors (BI-D1870 that is a RSK2 inhibitor, and 666-15 that is a CREB1 inhibitor) at 0.1-1 microM concentration. We evaluated viability by MTT assay or Zombie dye staining on CD138 positive MM cells and apoptosis by Annexin V-PI staining. We demonstrated that CD56 positive MM cell lines (H929, OPM-2, and RPMI-8226) or patients with high CD56 expression (>30% of CD56-expressing clonal MM cells) are more sensitive to RSK2/CREB1 inhibition compared with CD56 negative MM cell lines (U266, L363, and MM.1S) or patients with low CD56 expression (<30% of CD56-expressing clonal MM cells). Using similar strategies, we also proved that CREB1 is essential to CD56-protumoral phenotype, since CREB1 inhibition reduces cellular growth and viability in CD56 overexpressing U266 cells. RSK2 and CREB1 inhibition mimic CD56 silencing with decrease of BCL2 and MCL1 mRNA and protein levels. Furthermore, we observed that CD56 signaling by CREB1 activation decreases CRBN expression, reducing responses to lenalidomide. Conversely, CREB1/RSK2 blockade rescued CRBN levels in CD56 positive MM cells and increased lenalidomide response. These results support the hypothesis that targeting CREB1 is hence a so far unexplored but potentially effective synthetic lethal strategy for CD56-expressing MM patients.In conclusion, our study defines an effective threshold for therapeutic intervention in CD56-expressing MM patients. Moreover, our data pioneer the use of CREB1/RSK2 inhibition in CD56-expressing MM cells, either as single agents or in combination with lenalidomide, suggesting that CD56 can be a prognostic and predictive factor of response in MM. DisclosuresNo relevant conflicts of interest to declare.

Immunomolecular evaluation of dihydroartemisinin effects on apoptosis in chronic lymphocytic leukemia cell lines

IntroductionDihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, has recently shown to induce apoptosis in many types of cancer cells. In this study, we aimed to determine the effects of DHA on apoptosis in human chronic lymphocytic leukemia (CLL) cell lines. MethodsThe cells were treated separately and combined by DHA and Fludurabine (FLU) during 24, 48 and 72 hours. The cell viabilities determined by XTT method. Following separate and combined treatment of IC50 concentrations of DHA and FLU to the cells during 24 hours, the cells were analyzed by flow cytometry to determine the effects on apopotis staining with AnnexinV FITC and PI. mRNA and protein expression levels of TCTP, Mcl-1, Bcl-2, Bax and Caspase-3 were analyzed to find out the molecular mechanisms of apoptosis by using quantitative real-time PCR and flow cytometric methods. ResultsTreatment with DHA alone or in combination with FLU induced apoptosis in a dose dependent manner in CLL cells. DHA alone was more effective than FLU alone or combined treatment with DHA and FLU. Our results suggest that Bcl-2 protein family member Bax was active in the apoptotic response of CLL cells after DHA treatment. Moreover, the apoptotic response induced by DHA was independent from the p53 mutation status of the CLL cells. ConclusionDHA might be a potential anti-cancer therapeutic for CLL.

Pharmacologic targeting of the P-TEFb complex as a therapeutic strategy for chronic myeloid leukemia

BackgroundThe positive transcription elongation factor b (P-TEFb) kinase activity is involved in the process of transcription. Cyclin-dependent kinase 9 (CDK9), a core component of P-TEFb, regulates the process of transcription elongation, which is associated with differentiation and apoptosis in many cancer types. Wogonin, a natural CDK9 inhibitor isolated from Scutellaria baicalensis. This study aimed to investigate the involved molecular mechanisms of wogonin on anti- chronic myeloid leukemia (CML) cells.Materials and methodsmRNA and protein levels were analysed by RT-qPCR and western blot. Flow cytometry was used to assess cell differentiation and apoptosis. Cell transfection, immunofluorescence analysis and co-immunoprecipitation (co-IP) assays were applied to address the potential regulatory mechanism of wogonin. KU-812 cells xenograft NOD/SCID mice model was used to assess and verify the mechanism in vivo.ResultsWe reported that the anti-CML effects in K562, KU-812 and primary CML cells induced by wogonin were regulated by P-TEFb complex. We also confirmed the relationship between CDK9 and erythroid differentiation via knockdown the expression of CDK9. For further study the mechanism of erythroid differentiation induced by wogonin, co-IP experiments were used to demonstrate that wogonin increased the binding between GATA-1 and FOG-1 but decreased the binding between GATA-1 and RUNX1, which were depended on P-TEFb. Also, wogonin induced apoptosis and decreased the mRNA and protein levels of MCL-1 in KU-812 cells, which is the downstream of P-TEFb. In vivo studies showed wogonin had good anti-tumor effects in KU-812 xenografts NOD/ SCID mice model and decreased the proportion of human CD45+ cells in spleens of mice. We also verified that wogonin exhibited anti-CML effects through modulating P-TEFb activity in vivo.ConclusionsOur study indicated a special mechanism involving the regulation of P-TEFb kinase activity in CML cells, providing evidences for further application of wogonin in CML clinical treatment.8cwZMBLp1zasMdEPaNEt3gVideo

MiRNA-Mediated Knock-Down of Bcl-2 and Mcl-1 Increases Fludarabine-Sensitivity in CLL-CII Cells.

Background:Over-expression of anti-apoptotic proteins such as Bcl-2 and Mcl-1 is associated with resistance to chemotherapeutic agents such as fludarabine. Moreover, an inverse relationship between miRNA-15a levels with Bcl-2 and Mcl-1 expression has been observed in CLL patients. In this study, the effect of miRNA-15a on apoptosis and sensitivity of the CLL cells to fludarabine was investigated. Methods:After treatments, the Mcl-1 and Bcl-2 expression levels were quantified by RT-qPCR. Trypan blue assay was used to explore the effects of miRNA-15a and fludarabine on cell proliferation. The cytotoxicity was measured using MTT assay and combination index analysis. Cell death was determined using cell death detection ELISA assay and caspase-3 activity assay Kits. Results:Results showed that miRNA-15a clearly decreased the mRNA levels of Bcl-2 and Mcl-1 in a time dependent manner, which led to CLL-II cell proliferation inhibition and enhancement of apoptosis (p < 0.05, relative to control). Transfection of the miRNA-15a synergistically reduced the cell survival rate and lowered the IC50 value of fludarabine. Furthermore, miRNA-15a significantly enhanced the apoptotic effect of fludarabine. Conclusions:Our data propose that suppression of Bcl-2 and Mcl-1 by miRNA-15a can effectively inhibit the cell proliferation and sensitize CLL cells to fludarabine. Therefore, miRNA-15a can be considered as a potential therapeutic target in CLL resistant patients.

Shikonin treatment ameliorates Lipopolysaccharides (LPS) induced acute liver failure in mice via regulating the miR-106/MCL1 and miR-34a/SIRT1/TP53 signaling

IntroductionThe treatment with shikonin (SKN) suppresses the expression of miR-106 and miR-34a. Furthermore, SIRT1 and MCL1 are targets of miR-34a and miR-106, respectively. In this study, we treated an animal model of ALF with high dose (1.0 mg/kg) and low dose (0.5 mg/kg) of SKN to investigate its effect on liver functions and signaling pathways of SKN/miR-106/MCL1 and SKN/miR-34a/SIRT1/TP53.Material and methodsALF animal model was established and the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed to evaluate the effects of different doses of SKN. TUNNEL was performed to assess hepatocyte apoptosis. Luciferase assay, RT-qPCR and Western blot analysis were performed to measure the relationship between miR-106, miR-34a, SIRT1 and MCL1.ResultsIn the ALF mice models, the administration of SKN decreased the levels of ALT and AST in a dose-dependent manner, along with a significantly decreased number of apoptotic hepatocytes. And SKN may protect liver during ALF via reducing the level of inflammation. Luciferase assay showed that the co-transfection of wild-type MCL1/SIRT1 and miR-106/miR-34a significantly decreased the luciferase activity of LO2 cells, thus indicating that MCL1 and SIRT1 are identified as targets of miR-106 and miR-34a, respectively, while SIRT1 could act as a regulator of TP53. Moreover, the expression of miR-106, miR-34a and TP53 was decreased over an increasing concentration of SKN, along with the increasing mRNA and protein levels of MCL1 and SIRT1.ConclusionsIn this study, we showed that SKN alleviated ALF in a dose-dependent manner via regulating the signaling pathways of SKN/miR-106/MCL1 and SKN/miR-34a/SIRT1/TP53.

The combination of C-Myc rearrangement and 1q21 gain is associated with poor prognosis in multiple myeloma

The prognostic value of chromosomal 1q21 gain in newly diagnosed multiple myeloma (NDMM) remains controversial. Add-on Myc aberrations may further worsen the outcome. To investigate whether specific genes located at the 1q21 region, such as myeloid cell leukemia 1 (Mcl-1), are involved in NDMM progression, we examined bone marrow cytogenetic abnormalities in 153 patients with NDMM by fluorescence in situ hybridization. Their response to treatment and survival was also analyzed. C-Myc and Mcl-1 expressions in bone marrow samples were analyzed by RT-PCR. The expression of Mcl-1 was evaluated in bone marrow sections by immunohistochemistry. MM cell lines were transfected with Mcl-1 siRNA. 1q21 gain was present in 55/153 (35.9%) patients and strongly associated with Myc rearrangement (31/153, 20.3%, P = 0.004). A positive correlation was observed between Myc and Mcl-1 mRNA levels in bone marrow cells from 47 patients (r = 0.57, P < 0.001). The combination of 1q21 gain and Myc rearrangement was associated with poorer overall survival than Myc rearrangement alone (16.8 vs. 27.9 months, P = 0.077) or 1q21 gain alone (16.8 vs. 60.7 months, P < 0.01). High Mcl-1 protein expression in bone marrow plasma cells was associated with Myc rearrangement. Mcl-1 silencing by siRNA inhibited Myc protein expression in three myeloma cell lines. Treatment with the small-molecule Mcl-1 inhibitor, UMI-77, produced similar results. Overall, the combination of Myc rearrangement and 1q21 gain was associated with particularly poor prognosis in patients with MM. Furthermore, our data are consistent with Mcl-1-dependent Myc protein activation.

Ruxolitinib induces apoptosis of human colorectal cancer cells by downregulating the JAK1/2-STAT1-Mcl-1 axis.

Under pathological conditions, the Janus kinase (JAK)/STAT signaling pathway can regulate the proliferation, differentiation and migration of tumor cells, including colorectal cancer (CRC). CRC is the third major types of cancer among males and the second among females worldwide. In China, CRC is the fifth common cancer among both males and females. Western blotting, flow cytometry, RNA interference, immunoprecipitation, xenografts models, and immunohistochemical staining were carried out to evaluate the possible mechanisms of acton of ruxolitinib. The present data suggested that ruxolitinib can suppress CRC cell proliferation by inducing apoptosis. Firstly, JAK1/2-STAT1 was identified as the target of ruxolitinib. Then, ruxolitinib downregulated myeloid cell leukemia-1 (Mcl-1) mRNA level and decreased its protein level, which enabled Bak to trigger CRC apoptosis. Furthermore, ruxolitinib exerted potent activity against CRC xenograft growth in vivo. High expression of phosphorylated STAT1 (S727) was also confirmed in 44 pairs of human colon carcinoma and adjacent normal tissues. Taken together, the results showed that ruxolitinib decreased JAK1/2-STAT1-Mcl-1 protein level and effectively suppressed CRC cell proliferation in vitro and in vivo. Therefore, ruxolitinib could be a promising anticancer agent for CRC treatment.

Inhibition of EGFR pathway promotes the cytotoxicity of ABT-263 in human leukemia K562 cells by blocking MCL1 upregulation

ABT-263 induces MCL1 upregulation in cancer cells, which confers resistance to the drug. An increased understanding of the mechanism underlying ABT-263-induced MCL1 expression may provide a strategy to improve its tumor-suppression activity. The present study revealed that ABT-263 reduced the turnover of MCL1 mRNA, thereby upregulating MCL1 expression in human K562 leukemia cells. Furthermore, ABT-263-induced EGFR activation promoted AGO2 phosphorylation at Y393 and reduced miR-125b maturation. Treatment with EGFR inhibitors mitigated MCL1 upregulation induced by ABT-263. Additionally, lithium chloride (LiCl) alleviated ABT-263-induced MCL1 upregulation through EGFR-AGO2 axis-modulated miR-125b suppression. Ectopic expression of dominant negative AGO2(Y393F) or transfection with miR-125b abolished ABT-263-induced upregulation of MCL1 mRNA and protein levels. Co-treatment with either EGFR inhibitors or LiCl collaboratively enhanced ABT-263 cytotoxicity, while MCL1 overexpression eliminated this synergistic effect. Collectively, our data reveal that ABT-263 increases EGFR-mediated AGO2 phosphorylation, which in turn suppresses miR-125b-mediated MCL1 mRNA degradation in K562 cells. The suppression of this signaling pathway results in the synergistic cytotoxic effect of EGFR inhibitors or LiCl and ABT-263.

DYRK1A suppression restrains Mcl-1 expression and sensitizes NSCLC cells to Bcl-2 inhibitors.

Objective: Mcl-1 overexpression confers acquired resistance to Bcl-2 inhibitors in non-small cell lung cancer (NSCLC), but no direct Mcl-1 inhibitor is currently available for clinical use. Thus, novel therapeutic strategies are urgently needed to target Mcl-1 and sensitize the anti-NSCLC activity of Bcl-2 inhibitors.Methods: Cell proliferation was measured using sulforhodamine B and colony formation assays, and apoptosis was detected with Annexin V-FITC staining. Gene expression was manipulated using siRNAs and plasmids. Real-time PCR and Western blot were used to measure mRNA and protein levels. Immunoprecipitation and immunofluorescence were used to analyze co-localization of dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) and Mcl-1.Results: Suppression of DYRK1A resulted in reduced Mcl-1 expression in NSCLC cells, whereas overexpression of DYRK1A significantly increased Mcl-1 expression. Suppression of DYRK1A did not alter Mcl-1 mRNA levels, but did result in an accelerated degradation of Mcl-1 protein in NSCLC cells. Furthermore, DYRK1A mediated proteasome-dependent degradation of Mcl-1 in NSCLC cells, and DYRK1A co-localized with Mcl-1 in NSCLC cells and was co-expressed with Mcl-1 in tumor samples from lung cancer patients, suggesting that Mcl-1 may be a novel DYRK1A substrate. We showed that combined therapy with harmine and Bcl-2 antagonists significantly inhibited cell proliferation and induced apoptosis in NSCLC cell lines as well as primary NSCLC cells.Conclusions: Mcl-1 is a novel DYRK1A substrate, and the role of DYRK1A in promoting Mcl-1 stability makes it an attractive target for decreasing Bcl-2 inhibitor resistance.

Abstract A021: Genipin enhanced apoptosis of gastric cancer by mitochondrial dysfunction and downregulation of Stat3/JAK2/Mcl-1 signaling pathway

Abstract Genipin is known as a compound derived from gardenia fruit extract. Although Genipin is known to have an anti-tumor effects in various cancers, its effect and mechanism are unknown in gastric cancer. In our studies, Genipin induced apoptosis in gastric cancer cells including AGS and MKN45 cells. Genipin reduced Mcl-1 mRNA levels and confirmed with transfection of Mcl-1 siRNA. Furthermore, We found that phospho Stat3 is regulated by Genipin through proteome profiler phosphor-kinase array. In addition, protein level of phosphor JAK2 decreased in Genipin treatment. The results elucidate that Stat3/JAK2/Mcl-1 signaling pathway was suppressed with Genipin treatment in gastric cancer cells. Mcl-1 is known to be closely related to the function of mitochondria. Genipin has been shown to result in the collapse of mitochondrial function such as mitochondrial membrane potential. In conclusion, Genipin induced apoptosis by suppressing Stat3/JAK2/Mcl-1 pathway and led to mitochondrial dysfunction. Our finding implies that a novel mechanism of anti-cancer effect of Genipin in gastric cancer can be identified. Citation Format: Min Jee Jo, Soyeon Jeong, Hye Kyeong Yun, Dae Yeong Kim, Bo Ram Kim, Jung Lim Kim, Yoo Jin Na, Seong Hye Park, Yoon A Jeong, Bu Gyeom Kim, Hassan Ashktorab, Duane T Smoot, Jun Young Heo, Jeongsu Han, Dae-Hee Lee, Sang Cheul Oh. Genipin enhanced apoptosis of gastric cancer by mitochondrial dysfunction and downregulation of Stat3/JAK2/Mcl-1 signaling pathway [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A021. doi:10.1158/1535-7163.TARG-19-A021