Downregulation Of c-Myc Research Articles

Downregulation of c-Myc and p21 expression and induction of S phase arrest by naphthalene diimide derivative in gastric adenocarcinoma cells

Naphthalene diimide (NDI) derivatives have been shown to exhibit promising antineoplastic properties. In the current study, we assessed the anticancer and anti-bacterial properties of di-substituted NDI derivative. The naphthalene-bis-hydrazimide, 1, negatively affected the cell viability of three cancer cell lines (AGS, HeLa and PC3) and induced S phase cell cycle arrest along with SubG0/G1 accumulation. Amongst three cell lines, gastric cancer cell line, AGS, showed the highest sensitivity towards the NDI derivative 1. Compound 1 induced extensive DNA double strand breaks causing p53 activation leading to transcription of p53 target gene p21 in AGS cells. Reduction in protein levels of p21 and BRCA1 suggested that 1 treated AGS cells underwent cell death due to accumulation of DNA damage as a result of impaired DNA damage repair. β-catenin downregulation and consequently decrease in levels of c-Myc may have led to 1 induced AGS cell proliferation inhibition.1 induced AGS cell S phase arrest was mediated through CylinA/CDK2 downregulation. The possible mechanisms involved in anticancer activity of 1 includes ROS upregulation, induction of DNA damage, disruption of mitochondrial membrane potential causing ATP depletion, inhibition of cell proliferation and downregulation of antiapoptotic factors ultimately leading to mitochondria mediated apoptosis. Further compound 1 also inhibited H. pylori proliferation as well as H. pylori induced morphological changes in AGS cells. These findings suggest that NDI derivative 1 exhibits two-pronged anticancer activity, one by directly inhibiting cancer cell growth and inducing apoptosis and the other by inhibiting H. pylori.

Hesperidin inhibits cell proliferation and induces mitochondrial-mediated apoptosis in human lung cancer cells through down regulation of β-catenin/c-myc

Abstract The aim of this study is to evaluate the mechanism of actions involved in the anticancer effect of hesperidin against cell proliferation and inducing apoptosis in human lung cancer A549 cells. MTT assay were performed, by treating the cells with different concentrations of hesperidin ranges from 0, 6.25, 12.5, 25, 50 and 100 μM for 0, 6, 12, 24, and 48 h. Hesperidin treatment significantly inhibited the cell viability based on concentration and time dependent manner, which shows optimal LC 50 effective, dose as 12.5 and 25 μM for 24 h and this LC50 doses and optimized time point used for further studies. Hesperidin treated cells significantly inhibited the cell proliferation through down regulation of c-myc, β-catenin, PCNA protein expressions. An additional studies also shows that hesperidin significantly increased the p53 and p21 tumor suppressor proteins which subsequently inhibited the cyclin dependent kinase 4 (cdk4) and cyclin D protein expressions. Further studies supported that restoring of tumor suppressor proteins by hesperidin treatment directly affects the Bcl-2/Bax ratio, which limits the Bcl-2 levels and increased the pro-apoptotic Bax protein levels. Mitochondrial transmembrane potential studies, increased levels of cytochrome c, APAF-1, caspase-3 and DNA fragmentation was observed in hesperidin treated cells which clearly indicates apoptosis induction. Taken together, these findings suggest the anticancer effect of hesperidin in human lung cancer A549 cells through up regulation of p53, which triggers the proliferation arrest and mitochondrial dependent apoptosis activation.

C‐Myc mediated upregulation of long noncoding RNA SNHG12 regulates proliferation and drug sensitivity in natural killer/T‐cell lymphoma

Nasal-type natural killer/T-cell lymphoma (NKTCL) is an aggressive malignancy with poor outcomes. The treatment of NKTCL requires intensive chemotherapy. Long noncoding RNAs (lncRNAs) have been implicated in many cancers, including NKTCL. The elucidation of the multidrug resistance (MDR) may greatly contribute to explore novel therapeutic strategies. Herein, we explored the roles and potential regulatory mechanism of lncRNAs small nucleolar RNA host gene 12 (SNHG12) in MDR of NKTCL. We found that SNHG12 was upregulated in NKTCL tissue sections, and its high expression was positively correlated with clinical grade of malignancy of NKTCL. c-Myc and SNHG12 expression was upregulated in NKTCL cell lines. c-Myc- and SNHG12 overexpression promoted proliferation and inhibited sensitivity to cisplatin (CDDP) in NK/T-cell lymphoma cell line YTS cells, and c-Myc and SNHG12-downregulation inhibited proliferation and enhanced sensitivity to CDDP in SNK-6 cells. Moreover, c-Myc- and SNHG12 overexpression increased Ki67 and P-gp expression in YTS cells, whereas c-Myc and SNHG12-downregulation reduced the Ki67 and P-gp expression in SNK-6 cells. Correlational analyses revealed that c-Myc expression was positively correlated with SNHG12 expression in NKTCL tissues. Mechanism research showed that SNHG12 was a direct transcriptional target of c-Myc and c-Myc promoted SNHG12 expression in NKTCL cell lines. Further research showed that SNHG12 overexpression reversed the effects of c-Myc downregulation on proliferation and sensitivity to CDDP in NKTCL cell lines. Taken together, our findings first report that c-Myc mediated upregulation of SNHG12 promotes proliferation and inhibits drug sensitivity in NKTCL, which provides new insights into the therapeutic target for NKTCL.

SF2523 inhibits human chondrosarcoma cell growth in vitro and in vivo

Developing novel therapeutic agents against chondrosarcoma is important. SF2523 is a PI3K-Akt-mTOR and bromodomain-containing protein 4 (BRD4) dual inhibitor. Its activity in human chondrosarcoma cells is tested. Our results show that SF2523 potently inhibited survival, proliferation and migration, and induced apoptosis activation in SW1353 cells and primary human chondrosarcoma cells. The dual inhibitor was yet non-cytotoxic to the primary human osteoblasts and OB-6 osteoblastic cells. SF2523 blocked Akt-mTOR activation and downregulated BRD4-regulated genes (Bcl-2 and c-Myc) in chondrosarcoma cells. It was more efficient in killing chondrosarcoma cells than other established PI3K-Akt-mTOR and BRD4 inhibitors, including JQ1, perifosine and OSI-027. In vivo, intraperitoneal injection of SF2523 (30 mg/kg) potently inhibited subcutaneous SW1353 xenograft tumor growth in severe combined immunodeficient mice. Akt-mTOR inhibition as well as Bcl-2 and c-Myc downregulation were detected in SF2523-treated SW1353 tumor tissues. In conclusion, targeting PI3K-Akt-mTOR and BRD4 by SF2523 potently inhibited chondrosarcoma cell growth in vitro and in vivo.

Inhibition of telomerase activity and SK-OV-3/DDP cell apoptosis by rhodium(III) and iron(III) complexes with 4′-(3-thiophenecarboxaldehyde)-2,2′:6′,2″-terpyridine

Two rhodium(III) and iron(III) complexes of 4′-(3-thiophenecarboxaldehyde)-2,2′:6′,2″-terpyridine (tpatpy): [Rh(tpatpy)Cl3]·CH3OH (tpatpy-Rh) and [Fe(tpatpy)Cl3] (tpatpy-Fe) have been prepared and characterized. Tpatpy-Rh (5.03 ± 1.15 μM) and tpatpy-Fe (18.02 ± 0.45 μM) exhibited greater cytotoxicity than the tpatpy ligand (70.11 ± 0.28 μM) and cisplatin (61.21 ± 1.39 μM) against SK-OV-3/DDP cells. In addition, tpatpy-Rh inhibited telomerase by down-regulation of c-myc and hTERT, as well as induced SK-OV-3/DDP cell arrest in the S phase.

Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis.

AIMTo investigate the effect of adipose-derived mesenchymal stem cells (ADMSCs) and their conditioned media (CM) on hepatocellular carcinoma (HCC) cell tumorigenesis.METHODSThe proliferation rate of HepG2 and PLC-PRF-5 HCC cancer cells was measured using the trypan blue exclusion method and confirmed using the cell-counting kit 8 (commonly known as CCK-8) assay. Apoptosis was detected by flow cytometry using annexin V-FITC. Protein and mRNA expression was quantified by ELISA and real time PCR, respectively. Migration and invasion rates were performed by Transwell migration and invasion assays. Wound healing was examined to confirm the data obtained from the migration assays.RESULTSOur data demonstrated that when co-culturing HCC cell lines with ADMSCs or treating them with ADMSC CM, the HCC cell proliferation rate was significantly inhibited and the apoptosis rate increased. The decreased proliferation rate was accompanied by an upregulation of P53 and Retinoblastoma mRNA and a downregulation of c-Myc and hTERT mRNA levels. More notably, ADMSCs and their CM suppressed the expression of the two important markers of HCC carcinogenicity, alpha-fetoprotein and Des-gamma-carboxyprothrombin. In addition, the migration and invasion levels of HepG2 and PLC-PRF-5 cells significantly decreased, potentially through increased expression of the tissue inhibitor metalloproteinases TIMP-1, TIMP-2 and TIMP-3.CONCLUSIONThese findings shed new light on a protective and therapeutic role for ADMSCs and their CM in controlling HCC invasiveness and carcinogenesis.

5-Aza-CdR promotes partial MGMT demethylation and modifies expression of different genes in oral squamous cell carcinoma

Treatment strategies for oral squamous cell carcinoma (OSCC) vary, depending on the stage of diagnosis. Surgery and radiotherapy are options for localized lesions for stage I patients, whereas chemotherapy is the main treatment for metastatic OSCC. However, aggressive tumors can relapse, frequently causing death. In an attempt to address this, novel treatment protocols using drugs that alter the epigenetic profile have emerged as an alternative to control tumor growth and metastasis. Therefore, the objective in this study was to investigate the effect of the demethylating drug 5-aza-CdR in SCC9 OSCC cells. SCC9 cells were treated with 5-Aza-CdR at concentrations of 0.3μM and 2μM for 24hours and 48hours. DNA methylation of the MGMT, BRCA1, APC, c-MYC, and hTERT genes were investigated by using the methylation-specific high-resolution melting technique. Real time-polymerase chain reaction and quantitative polymerase chain reaction were performed to analyze gene expression. 5-Aza-CdR promoted demethylation of MGMT and modified the transcription of all analyzed genes. Curiously, 5-aza-CdR at the concentration of 0.3μM was more efficient than 2μM in SCC9 cells. We observed that 5-aza-CdR led to MGMT demethylation, upregulated the transcription of 3 important tumor suppressor genes, and promoted the downregulation of c-Myc.

Whole-exome sequencing reveals novel genetic variants associated with diverse phenotypes of melanoma cells.

We have extensively studied the phenotypic heterogeneity of patient-derived melanoma cells. Here, whole-exome sequencing revealed novel variants of genes associated with the MAPK, NOTCH, Hippo, cell-cycle, senescence, and ubiquitin-dependent pathways, which could contribute to the observed phenotypic diversity between cell lines. Focusing on mutations in the MAPK pathway-associated genes, we found BRAF (BRAFV600E ) and RAS subtypes, including NRASQ61R and the rare HRASQ61R variant, and additional alterations potentially leading to different ERK1/2 activity. Both RASQ61R cell lines harbored a MEK1P124S variant and exerted a low level of phospho-MEK1/2. Activity of the MAPK pathway was further attenuated in NRASQ61R /MEKP124S cells by trametinib, and this effect was also shown in HRASQ61R /MEKP124S melanoma cells. The observed variability in doubling time might be a consequence of diverse MAPK and PI3K/AKT pathway activities, but not exclusively, as a senescence program was also executed to different extent in distinct melanoma cell lines. Low percentages of senescent cells might result from mutations in CDKN2A, E2F3, and EZH2, and a high c-MYC expression. Vemurafenib and trametinib induced senescence concomitantly with c-MYC downregulation and irrespectively of CDKN2A mutation, but the EZH2S412C variant might limit senescence induction. Damaging alterations in Hippo pathway-associated genes were accompanied with variability in the phosphorylation level of YAP1/TAZ and CTGF expression. Our study also suggests opposite activity of NOTCH2F1209V and NOTCH2N2002S variants. Additionally, we found a novel FBXW7V418M variant that retained its function in melanoma cells. The obtained molecular data might be further exploited in genotype-phenotype relationship studies and in identifying novel biomarkers and therapies for melanomas.

Curcumin combining with si-MALAT1 inhibits the invasion and migration of colon cancer SW480 cells

To study the effect of small interfering RNA targeting metastasis-associated lung adenocarcinoma transcript1 (si-MALAT1) combining with curcumin on the invasion and migration abilities of human colon cancer SW480 cells, and to explore the involved molecular mechanism. The recombinant lentiviral vector expressing si-MALAT1 was constructed, and its titer was determined by gradient dilution method. The colon cancer SW480 cells with stable expression of si-MALAT1 was established, followed by treatment with curcumin at different concentrations. The effect of curcumin or si-MALAT1 alone and the combination of the two on the cell activity was detected by MTT assay. The cell invasion and migration abilities were detected by transwell and scratch-wound assay. The relative expression level of MALAT1 was detected by RT-qPCR. The protein expression was determined by Western blot analysis. The IC50 of curcumin alone was 77.69 µmol/L, which was 51.17 mol/Lwhen combined with curcumin and random sequence. The IC50 of curcumin was 30.02 µmol/L when combined with si-MALAT1. The increased susceptibility multiples was 2.58. The wound healing rates were 30.9% and 67.5% after treatment with si-MALAT1 combined with curcumin for 24 hrs and 48 hrs, respectively. The numbers of invasion cells were 200±12, 162±13, 66±8, 53±4 and 16±3 after treatment with si-MALAT1 combined with curcumin for 48 hrs. The relative expression level of lncRNA-MALAT1 in the curcumin group was 68%, and the relative expression level of lncRNA-MALAT1 in si-MALAT1group was 56%, and that for the combination treatment group was about 21%. The protein expression levels of β- catenin, c-myc and cyclinD1 were significantly down-regulated upon treatment with certain concentration of si-MALAT1 alone or combined with curcumin.si-MALAT1 could significantly inhibit the invasion and migration of SW480 cells by enhancing the sensitivity of SW480 cells to curcumin. The mechanism involved mignt be related to the down-regulation of β-catenin, c-myc and cyclinD1 proteins.

Erwinia Chrysantemi-Derived L-Asparaginase Strongly Enhances Proteasome Inhibitors Activity By Deregulating Cell Metabolic Programs

Background: Human tumours rely on Glutamine (Gln) metabolism to sustain wide range of metabolic processes and macromolecules synthesis. Such dependence defined as “Gln-addiction” has been recently observed also for Multiple Myeloma (MM) cells, where Gln synthetase deficiency make them more sensitive to Gln depletion. L-Asparaginase (L-ASP), the enzyme that catalyzes asparagine (Asn) and Gln hydrolysis, represents a key drug for B-cells derived malignancies treatment such as acute lymphoblastic leukemia.Aims: The aim of the present study was to screen MM cells sensitivity to pharmacological Gln depletion achieved by Erwinia chrysantemi-derived L-ASP treatment as single agent as well as in combination with proteasome inhibitors (PIs). Based on its high glutaminase activity, E. chrysantemi L-ASP was selected for this screening.Materials and methods: The IC50 value of L-ASP was determined by MTS assay in a panel of human MM cell lines (HMCLs) harboring different genetic background as well as on primary CD138+ cells derived from newly diagnosed (NDMM) and and relapsed/refractory (RRMM) MM patients. Cell death analysis was measured with Annexin V/Propidium Iodide (AV/PI) staining followed by flow-cytometric analysis. L-ASP treatment effect on cell viability and mitochondrial membrane potential was evaluated by FACS analysis following PI and TMRE staining, respectively. Intracellular ATP levels was also measured by HPLC-based method. Drugs combination screening with L-ASP plus PIs including bortezomib (BZ) or carfilzomib (KAR) was investigated by using isobologram analysis (CalcuSyn software program). Finally, western blot analysis was employed to fully elucidate biological mechanisms of our findings.Results: L-ASP treatment showed potent anti-MM activity in all HMCLs tested with an IC50 values ranging from 0,03 U/mL to 0.2 U/mL. Importantly, such drug preserved its effect also on primary tumor cells obtained from NDMM and RRMM patients (IC50 =0.06 U/mL). Cell cycle analysis revealed an early (24 h) but irreversible “metabolic shutdown” in MM cells treated with L-ASP which led to late mitochondrial stress (48-72h). To further confirm Gln specific role in the observed finding, MM cells viability was measured in presence of different culture media conditions. Asn depletion alone did not modify significantly MM cells viability, instead it strongly contributed to L-ASP induced toxicity of Gln-starved cells. Co-treatment experiments showed massive increase of its anti-MM activity when L-ASP was used in presence of low doses of BZ and KAR, with a combination index below than 1 in all tested drugs-concentrations. Accordingly, caspase-3 and PARP cleavage showed apoptotic cell death features of this co-treatment. Importantly, IL6 or IGF-1 addition did not reduce anti-MM activity of this combination suggesting that MM microenvironment does not reduce efficacy of this strategy. Finally, mechanistic studies revealed higher ATP shortage in co-treated cells together with c-MYC down-regulation, genomic instability and misfolded proteins accumulation, suggesting a massive proteasome activity impairment triggered by these drugs-combination. Ongoing experiment will help to further elucidate this hypothesis.Conclusions: Our preliminary data show that E. chrysantemi L-ASP exerts a potent anti-MM activity mainly due to its glutaminase activity, which results in a massive metabolic program deregulation. The combination with low-doses of PIs further increases anti-MM activity of this small molecule likely due to huge proteasome activity injury. DisclosuresGobbi:Amgen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Janssen: Consultancy; Pfister: Membership on an entity's Board of Directors or advisory committees.

Concurrent FLT3 Inhibitor and PP2A Activating Drug Treatment Induces Synergistic Cytotoxicity in Acute Myeloid Leukemia Cells with FLT3 Internal Tandem Duplication through Proteasomal Degradation of Pim-1 and c-Myc

Introduction: In 30% of acute myeloid leukemia (AML) patients, internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) causes constitutive and aberrant FLT3 signaling, and these patients have short relapse-free and overall survival. FLT3 inhibitors have limited and transient efficacy, but their efficacy may be enhanced by combination with other drugs targeting FLT3 signaling. FLT3 activation also inhibits the tumor suppressor protein phosphatase 2A (PP2A). FLT3 inhibitors and PP2A-activating drugs have been shown to induce synergistic cytotoxicity in cells with FLT3-ITD. To address mechanisms underlying this effect, we studied effects of combination therapy on the oncogenic serine/threonine kinase Pim-1 and the transcription factor c-Myc, both of which are upregulated in cells with FLT3-ITD and are also PP2A substrates.Methods: Ba/F3-ITD and MV4-11 cells and AML patient blasts with FLT3-ITD were cultured with a FLT3 inhibitor, gilteritinib (ASP2215) or quizartinib (AC220), and/or the PP2A-activating drug fingolimod (FTY720) at pharmacologically relevant concentrations, or DMSO control. Drug combination effects were measured by combination index determined by the Chou-Talalay method using CompuSyn software. Apoptosis was measured by Annexin V/propidium iodide staining detected by flow cytometry. c-Myc and GAPDH control mRNA was measured by real-time polymerase chain reaction. Pim-1 kinase, c-Myc, phospho-c-MycSer62, phospho-c-MycThr58, phospho-STAT5Tyr694, STAT5, phospho-PP2ATyr307, PP2A, phospho-BADSer112 and BAD levels were measured by immunoblotting. Cycloheximide treatment was used to assess protein stability. Protein expression and stability were measured with and without the proteasome inhibitor MG-132. Pim-1 kinase was inhibited with the pan-Pim inhibitor AZD1208. Ba/F3-ITD cells were infected with pMX-Flag-K67M kinase-dead (KD) Pim-1 and empty pMX retroviral vectors and with pBABE-ER-cMYC and with empty pBABE-ER retroviral vectors.Results: Concurrent treatment with 15 nM gilteritinib or 1 nM quizartinib and FTY720 2 µM in cell lines and 4 µM in patient samples decreased growth and increased apoptosis of cells with FLT3-ITD, relative to single drug treatments, and produced synergistic cytotoxicity. FLT3 inhibition was confirmed by decrease in phospho-STAT5 and PP2A activation by decreased phospho-PP2A. Concurrent treatment decreased expression of both Pim-1 and c-Myc protein, but not c-Myc mRNA, in Ba/F3-ITD and MV4-11 cells and AML patient blasts with FLT3-ITD, relative to single drug treatments. Additionally, selective decrease in phospho-MycSer62, a stable c-Myc phosphoprotein that is dephosphorylated by PP2A, was seen, with persistence of phospho-c-MycThr58. FLT3 inhibitor and PP2A activator combination treatment was found to decrease stability of c-Myc and Pim-1 protein, in relation to single drugs. Moreover, pretreatment with the proteasome inhibitor MG-132 abrogated downregulation of Pim-1 and c-Myc protein expression and decrease in Pim-1 and c-Myc protein stability in Ba/F3-ITD cells treated with FLT3 inhibitor and PP2A activator. Pretreatment with the pan-Pim kinase inhibitor AZD1208, with Pim-1 inhibition confirmed by decreased phospho-BADS112 had no effect on c-Myc downregulation, and c-Myc was similarly downregulated in Pim-1 kinase-dead cells as in parental and empty-vector cells, demonstrating that combination treatment effects on c-Myc are not Pim-1 kinase-dependent. Additionally, FLT3 inhibitor and PP2A-activating drug combination induced apoptosis in 30% of cells with c-Myc overexpression, compared to 60% of parental and empty vector-infected cells. Finally, c-Myc overexpression did not abrogate Pim-1 downregulation by combination treatment.Conclusions: Concurrent FLT3 inhibitor and PP2A activating drug treatment induces synergistic cytotoxicity in AML cells with FLT3 internal tandem duplication through proteasomal degradation of Pim-1 and c-Myc, and effects on Pim-1 and c-Myc are independent. The data support in vivo testing of FLT3 inhibitor and PP2A-activating drug combinations and development of a clinical trial. DisclosuresNo relevant conflicts of interest to declare.

Lncrna RP1-68D18.2 Promotes K562 Cell Line Resistance to Imatinib through CD44 and Its Downstream Signal Molecules

As the first-generation tyrosine kinase inhibitor specifically targeting the BCR-ABL oncoprotein, imatinib has been successfully used in the therapy for chronic myelogenous leukemia (CML) with a significant response and good compliance. However, imatinib resistance becomes the main reason for failure of the treatment of CML. In addition to the common BCR-ABL dependence of imatinib resistance, e.g. BCR-ABL kinase domain mutations and BCR-ABL gene amplification, BCR-ABL independence also plays important roles, which involves alteration of dynamics of drug import and efflux, persistent or compensatory activation of signaling pathways etc. Recently, several studies have reported the correlation of lncRNA with resistance of chemotherapeutic drugs in many cancers. Here, we report, for the first time, discernible overexpression of lncRNARP1-68D18.2 and CD44 mRNA in the imatinib resistant chronic myelogenous leukemia cell line K562R, using lncRNA and mRNA microarray, and further verify it using qPCR. Knockdown of RP1-68D18.2 in K562R cell lines increased their sensitivity to imatinib, and simultaneously caused a decrease in CD44 and c-Myc protein levels. Correspondingly, overexpression of RP1-68D18.2 in K562, a sensitive cell line, led to a slightly decrease in imatinib sensitivity and partially reverse downregulation of c-Myc, p-ERK and p-MEK caused by imatinib treatment. Analysis of correlations revealed positive correlation between RP1-68D18.2 and CD44 mRNA expression in the primary CML blasts and hematological malignant cell lines. We conclude that overexpression of RP1-68D18.2 could promote imatinib resistance in the K562R cell line through the regulation of CD44 and its downstream signal molecules. DisclosuresNo relevant conflicts of interest to declare.

Lupeol inhibits growth and migration in two human colorectal cancer cell lines by suppression of Wnt-β-catenin pathway.

BackgroundLupeol, a triterpene isolated from various herbal plants, possesses an anti-inflammatory function and has been proposed as a candidate for anticancer agents. The purpose of this research was to investigate the effect of lupeol on the viability, apoptosis, cell-cycle distribution, and migration of colorectal cancer cell lines and its molecular mechanism.MethodsLupeol was assessed for its anticancer effect using two human colorectal cancer cell lines: SW480 and HCT116. These cells were treated with lupeol, and their viability, apoptosis, migration, and cycle distribution were detected by CCK8, flow cytometry, and the transwell method. Quantitative PCR, Western blot, and immunofluorescence were applied to detect the expressions of CTNNB1, TCF4, cMYC, CCND1, CLDN1, and CCNA2.ResultsLupeol suppressed cell viability and migration and induced cellular apoptosis of both cell lines, with increased p53 and decreased Bcl2 protein levels (P<0.05). Cell cycles of both lupeol-treated cell lines were arrested in the S phase (P<0.05). Quantitative PCR and Western blot analyses showed significantly reduced expressions of CTNNB1, TCF4, and downstream genes of the Wnt–β-catenin pathway, including the cell-cycle-regulated genes of cMYC and CCND1 of both cell lines upon lupeol treatment (P<0.05). mRNA and protein levels of CLDN1 decreased in HCT116 cells, plus the expression of CCNA2 mRNA and protein decreased in SW480 cells (P<0.05). Immunofluorescence analysis confirmed decreased expression of Wnt–β-catenin signaling.ConclusionOur findings indicate that lupeol effectively inhibits proliferation and migration and induces apoptosis and cell-cycle arrest of two colorectal cell lines by inactivation of the Wnt–β-catenin signaling pathway and downregulation of cMYC, CCND1, CCNA2, and CLDN1, thereby making it a promising anticancer candidate.

Diallyl disulfide suppresses FOXM1-mediated proliferation and invasion in osteosarcoma by upregulating miR-134.

Diallyl disulfide (DADS), a volatile component of garlic oil, exerts anticancer activity in various types of cancers, while its anticancer effects against osteosarcoma (OS) have not been previously explored. This study aimed to investigate the anticancer potential of DADS in OS and to explore the underlying mechanisms. DADS reduced the cell viability and increased the expression of miR-134 in OS cell lines, and this effect was in a time- and concentration-dependent manner. Furthermore, in vitro functional assays revealed that DADS significantly inhibited the proliferation and invasion of human OS U2OS and MG-63 cells, which was partially reversed by miR-134 inhibitor transfection. DADS exhibited in vivo antitumor activity and upregulated miR-134 expression in xenograft tumors. Downregulation of miR-134 attenuated DADS-induced antitumor capacity. Further bioinformatics prediction analysis revealed that the 3'-untranslated region (3'-UTR) of Forkhead Box M1 (FOXM1) harbored miR-134-binding sites, and overexpression of miR-134 repressed the luciferase activity of the reporting vector containing FOXM1 3'-UTR. Both miR-134 overexpression and DADS inhibited FOXM1 expression in U2OS cells, while enforced expression of FOXM1 suppressed DADS-induced antiproliferation and anti-invasion capacity in U2OS cells. Furthermore, DADS treatment led to significant downregulation of cyclin D1, c-myc, and lymphoid enhancer-binding factor 1 expression, but the remarkably upregulated p21 level in U2OS cells. Collectively, DADS could be a promising anticancer agent for OS, and the underlying mechanisms might be associated with the antiproliferation and anti-invasion properties through upregulating miR-134 expression.

The Myc/Max/Mxd Network Is a Target of Mutated Flt3 Signaling in Hematopoietic Stem Cells in Flt3-ITD-Induced Myeloproliferative Disease.

Acute myeloid leukemia (AML) has poor prognosis due to various mutations, e.g., in the FLT3 gene. Therefore, it is important to identify pathways regulated by the activated Flt3 receptor for the discovery of new therapeutic targets. The Myc network of oncogenes and tumor suppressor genes is involved in mechanisms regulating proliferation and survival of cells, including that of the hematopoietic system. In this study, we evaluated the expression of the Myc oncogenes and Mxd antagonists in hematopoietic stem cell and myeloid progenitor populations in the Flt3-ITD-knockin myeloproliferative mouse model. Our data shows that the expression of Myc network genes is changed in Flt3-ITD mice compared with the wild type. Mycn is increased in multipotent progenitors and in the pre-GM compartment of myeloid progenitors in the ITD mice while the expression of several genes in the tumor suppressor Mxd family, including Mxd1, Mxd2, and Mxd4, is concomitantly downregulated, as well as the expression of the Mxd-related gene Mnt and the transcriptional activator Miz-1. LSKCD150+CD48− hematopoietic long-term stem cells are decreased in the Flt3-ITD cells while multipotent progenitors are increased. Of note, PKC412-mediated inhibition of Flt3-ITD signaling results in downregulation of cMyc and upregulation of the Myc antagonists Mxd1, Mxd2, and Mxd4. Our data provides new mechanistic insights into downstream alterations upon aberrant Flt3 signaling and rationale for combination therapies for tyrosine kinase inhibitors with Myc antagonists in treating AML.

CUDC-907 displays potent antitumor activity against human pancreatic adenocarcinoma in vitro and in vivo through inhibition of HDAC6 to downregulate c-Myc expression.

Pancreatic adenocarcinoma is a highly malignant cancer that often involves a deregulation of c-Myc. It has been shown that c-Myc plays a pivotal role in the regulation of a variety of physiological processes and is involved in early neoplastic development, resulting in poor progression. Hence, suppression of c-Myc overexpression is a potential strategy for pancreatic cancer therapy. CUDC-907 is a novel dual-acting inhibitor of phosphoinositide 3-kinase (PI3K) and histone deacetylase (HDAC). It has shown potential efficiency in patients with lymphoma, multiple myeloma, or thyroid cancer, as well as in solid tumors with c-Myc alterations, but the evidence is lacking for how CUDC-907 regulates c-Myc. In this study, we investigated the effect of CUDC-907 on human pancreatic cancer cells in vitro and in vivo. Our results showed that CUDC-907 potently inhibited the proliferation of 9 pancreatic cancer cell lines in vitro with IC50 values ranging from 6.7 to 54.5 nM. Furthermore, we revealed the antitumor mechanism of CUDC-907 in Aspc-1, PANC-1, and Capan-1 pancreatic cancer cells: it suppressed the HDAC6 subunit, thus downregulating c-Myc protein levels, which was a mode of action distinct from the existing mechanisms. Consistently, the extraordinary antitumor activity of CUDC-907 accompanied by downregulation of c-Myc and Ki67 expression in tumor tissue was observed in a human pancreatic cancer Aspc-1 xenograft nude mouse model in vivo. Our results suggest that CUDC-907 can be a valuable therapeutic option for treating pancreatic adenocarcinoma.

Impaired mammary tumor formation and metastasis by the point mutation of a Smad3 linker phosphorylation site

Triple-negative breast cancer (TNBC) is often aggressive and metastatic. Transforming growth factor-β acts as a tumor-promoter in TNBC. Smad3, a major downstream effector protein in the TGF-β signaling pathway, is regulated by phosphorylation at several sites. The functional significance of the phosphorylation of the linker region in Smad3 is poorly understood for TNBC. Among the four sites in the Smad3 linker region, threonine-179 (T179) appears to be unique as it serves as the binding site for multiple WW-domain-containing proteins upon phosphorylation, suggesting that this phosphorylation is a key for Smad3 to engage other pathways.Using genome editing, we introduced for the first time a knock-in (KI) mutation in the endogenous Smad3 gene in IV2, a lung-tropic subline of the human MDA-MB-231 TNBC cell line. In the resulting cell line, the Smad3 T179 phosphorylation site is replaced by non-phosphorylatable valine (T179V) with the mutation in both alleles.The T179V KI reduced cell growth rate and mammosphere formation. These phenomena were accompanied by a significant upregulation of p21Cip1 and downregulation of c-Myc. The T179V KI also reduced cell migration and invasion in vitro. In the mouse xenograft models, the T179V KI markedly reduced the establishment of primary tumor in the mammary fat pad and the lung metastasis.Our results using gene editing indicate the cancer-promoting role of Smad3 T179 phosphorylation in the human TNBC cells. Our findings highly suggest that controlling this phosphorylation may have therapeutic potential for TNBC.

Knockdown of Long Noncoding RNA Plasmacytoma Variant Translocation 1 with Antisense Locked Nucleic Acid GapmeRs Exerts Tumor-Suppressive Functions in Human Acute Erythroleukemia Cells Through Downregulation of C-MYC Expression.

Objective: Acute erythroleukemia (AEL) is a subtype of acute myeloid leukemia (AML), with no specific treatment. Up- or downregulation of long noncoding RNAs (lncRNAs) is strongly associated with the formation and progression of many malignancies. Plasmacytoma variant translocation 1 (PVT1) is a significantly upregulated lncRNA in AML. Antisense locked nucleic acid (LNA) GapmeRs oligonucleotides are the novel tools for targeting lncRNAs. The purpose of the current study was to investigate the functional role of PVT1 antisense LNA GapmeRs on AEL cell line (KG-1). Materials and Methods: AEL cells were transfected with PVT1 antisense LNA GapmeRs at three different time points. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was accomplished to evaluate the PVT1 expression by PVT1 antisense LNA GapmeRs. The viability was evaluated by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) assay, and the apoptosis and necrosis were assessed by Annexin V/propidium iodide staining assay. The C-MYC expression level, the target gene of PVT1, was also quantified by qRT-PCR. Results: The results indicated that PVT1 inhibition could significantly decrease the viability of AEL cells, due to induction of apoptosis and necrosis, probably through the downregulation of C-MYC. Conclusions: Their findings suggest that the inhibition of lncRNA PVT1 could serve as a novel approach for controlling the proliferation of AEL cells and could open up a path for treatment of AEL.

Iodine‑125 interstitial brachytherapy reduces tumor growth via Warburg effect inhibition in non‑small cell lung cancer A549 xenografts

Iodine-125 interstitial brachytherapy (125I-IBT) is an alternative and effective treatment option for unresectable non-small cell lung cancer (NSCLC), and the Warburg effect is a determinant of tumor growth. The present study aimed to explore the influence of 125I-IBT on tumor growth and the Warburg effect, and the potential mechanisms underlying NSCLC progression. Mice with A549 cell xenografts were evenly divided into a control group without 125I-IBT, and three treatment groups receiving 125I-IBT with 20, 40 and 60 Gy. Tumor volume (TV), maximum standardized uptake value (SUVmax) determined by 18F-fluorodeoxyglucose (18F-FDG) micro-positron emission tomography/computed tomography and mean optical density (MOD) of mammalian target of rapamycin (mTOR), c-Myc, hypoxia inducible factor-1α (HIF-1α) and glucose transporter 1 (GLUT1) staining were compared among groups. Tumor inhibition rate (TIR), 18F-FDG uptake attenuation rate (FUAR) and expression suppression rate (ESR) were also calculated on day 14 and 28. The results demonstrated that the mean TV in the 60 and 40 Gy groups was smaller compared with the control TVs since days 14 and 16, respectively. The mean SUVmax value of the 60 Gy group at day 14, and all treatment group SUVmax values at day 28 were lower compared with the controls. In addition, the MOD of mTOR and GLUT1 was lower in the 60 Gy group, compared with the other groups, and c-Myc and HIF-1α values were lower in the 40 and 60 Gy groups, compared with the control and 20 Gy group (P<0.05). SUVmax positively correlated to TV (day 14, r=0.711; day 28, r=0.586) and the MOD of c-Myc and GLUT1 (r=0.621 and 0.546, respectively; P<0.01). Furthermore, dose dependent increases were observed for TIR, FUAR and ESR. In conclusion, 125I-IBT reduced tumor growth by inhibiting the Warburg effect, which may have resulted from downregulation of mTOR, c-Myc, HIF-1α and GLUT1 expression, particularly c-Myc and GLUT1, in NSCLC A549 ×enografts.

4-Acetyl-Antroquinonol B Suppresses SOD2-Enhanced Cancer Stem Cell-Like Phenotypes and Chemoresistance of Colorectal Cancer Cells by Inducing hsa-miR-324 re-Expression.

Background: Colorectal cancer (CRC) remains a leading cause of cancer-related morbidity and mortality in both sexes globally. This is not unconnected with the heterogeneity and plasticity of CRC stem cells (CRC-SCs) which stealthily exploit the niche-related and (epi)genetic factors to facilitate metastasis, chemoresistance, tumor recurrence, and disease progression. Despite the accumulating evidence of the role of dysregulated microRNAs in malignancies, the therapeutic efficacy of pharmacological-targeting of CRC-SC-associated microRNAs is relatively under-explored. Experimental approach: In this present study, we employed relatively new bioinformatics approaches, analyses of microarray data, Western blot, real-time polymerase chain reaction (RT-PCR), and functional assays to show that hsa-miR-324-5p expression is significantly suppressed in CRC cells, and inversely correlates with the aberrant expression of SOD2. Results: This converse hsa-miR-324-5p/SOD2 relationship is associated with enhanced oncogenicity, which is effectively inhibited by 4-acetylantroquinonol B (4-AAQB), as evidenced by inhibited cell viability and proliferation, as well as attenuated migration, invasion, and clonogenicity in 4-AAQB-treated DLD1 and HCT116 cells. Interestingly, 4-AAQB did not affect the viability and proliferation of normal colon cells. We also showed that 4-AAQB-induced re-expression of hsa-miR-324-5p, akin to short-interfering RNA, reduced SOD2 expression, correlates with the concurrent down-regulation of SOD2, N-cadherin, vimentin, c-Myc, and BcL-xL2, with concomitant up-regulation of E-cadherin and BAX2 proteins. Enhanced expression of hsa-miR-324-5p in the CRC cells suppressed their tumorigenicity in vitro and in vivo. Additionally, 4-AAQB synergistically potentiates the FOLFOX (folinate (leucovorin), fluorouracil (5FU), and oxaliplatin) anticancer effect by eliciting the re-expression of SOD2-suppressed hsa-miR-324, and inhibiting SOD2-mediated tumorigenicity. Conclusion: Our findings highlight the pre-clinical anti-CSC efficacy of 4-AAQB, with or without FOLFOX in CRC, and suggest a potential novel therapeutic strategy for CRC patients.