Limited Antiproliferative Effects Research Articles

Variation of bioactive potentials during the production of enzyme-modified cheese

Milk proteins are a known source of bioactive peptides released through proteolysis. Enzyme-modified cheese (EMC), produced with high proteolysis levels, has the potential to provide such peptides. This study aimed to explore the bioactive potentials (antioxidant, antihypertensive, antidiabetic, antimicrobial, and antiproliferative potentials) of EMC during production stages and after in vitro digestion. Results showed remarkable increases in antioxidant activities and sustained ACE inhibition activity values with each production stage. The samples inhibited α-amylase activity before in vitro digestion, while significant inhibition of α-glucosidase activity was observed after digestion. Furthermore, antimicrobial effects on Staphylococcus aureus were observed after digestion, with limited antiproliferative effects on various cancer cells. The study demonstrates the potential of EMC to positively impact body functions through its bioactive properties. Further research is needed to investigate the components formed during the production stages in detail.

Amivantamab (JNJ-61186372), an Fc Enhanced EGFR/cMet Bispecific Antibody, Induces Receptor Downmodulation and Antitumor Activity by Monocyte/Macrophage Trogocytosis.

Small molecule inhibitors targeting mutant EGFR are standard of care in non-small cell lung cancer (NSCLC), but acquired resistance invariably develops through mutations in EGFR or through activation of compensatory pathways such as cMet. Amivantamab (JNJ-61186372) is an anti-EGFR and anti-cMet bispecific low fucose antibody with enhanced Fc function designed to treat tumors driven by activated EGFR and/or cMet signaling. Potent in vivo antitumor efficacy is observed upon amivantamab treatment of human tumor xenograft models driven by mutant activated EGFR, and this activity is associated with receptor downregulation. Despite these robust antitumor responses in vivo, limited antiproliferative effects and EGFR/cMet receptor downregulation by amivantamab were observed in vitro Interestingly, in vitro addition of isolated human immune cells notably enhanced amivantamab-mediated EGFR and cMet downregulation, leading to antibody dose-dependent cancer cell killing. Through a comprehensive assessment of the Fc-mediated effector functions, we demonstrate that monocytes and/or macrophages, through trogocytosis, are necessary and sufficient for Fc interaction-mediated EGFR/cMet downmodulation and are required for in vivo antitumor efficacy. Collectively, our findings represent a novel Fc-dependent macrophage-mediated antitumor mechanism of amivantamab and highlight trogocytosis as an important mechanism of action to exploit in designing new antibody-based cancer therapies.

Abstract 4691: Identification and characterization of BRPF1 bromodomain inhibitors

Abstract BRPF (bromodomain and PHD finger-containing) proteins represent a subgroup of the bromodomain protein family with three closely related members, namely BRPF1, -2, and -3. They are found in different histone acetyltransferase complexes and regulate development and cell differentiation. Despite their high similarity, they have unique, non-redundant functions. Knock-out of BRPF1 in mice leads to embryonic lethality at E9.5 due to vascular defects whereas BRPF2 deficiency leads to embryonic lethality at E15.5 due to anemia. BRPF proteins contain multiple functional regions including a PZP domain which binds to unmodified H3K4, a PWWP domain which binds to H3K36me3 and a single bromodomain which recognizes acetyl-lysines in histones. BRPF1 is overexpressed in a variety of tumors, suggesting a role in cell proliferation. As first steps towards understanding the role of BRPF1, we performed knock-down studies in different tumor cell lines and initiated the search for potent and selective inhibitors of the BRPF1 bromodomain. BRPF1-specific siRNAs were designed and used for gene silencing experiments in bladder and ovarian cancer cell lines. Following BRPF1 knock-down, marked inhibition of proliferation was observed in the CAL-29, 5637 and JMSU-1 bladder cancer cell lines, and in the ES-2 and OVCAR-8 ovarian cancer cell lines. In order to further delineate the function of BRPF1, we sought to identify selective BRPF1 bromodomain inhibitors. A collection of 5000 compounds was selected by virtual screening based on the BRPF1 bromodomain crystal structure (4LC2) and tested in a biochemical TR-FRET assay for inhibition of the interaction between BRPF1 bromodomain and an acetylated histone H4-derived peptide. Two potent hit clusters (IC50 < 150 nM) were identified and subsequent optimization led to BAY-140 and BAY-496 which were selected for further analyses. The IC50 values of BAY-140 and BAY-496 for inhibition of BRPF1 bromodomain binding were 30 and 20 nM, and for BRPF2 bromodomain binding 380 and 160 nM, respectively. Importantly, no interaction was seen for either BRD4 bromodomain (IC50 >20 μM). This binding and selectivity profile was confirmed in cellular NanoBRET assays where IC50 values for BAY-140 and BAY-496 were 1300 and 810 nM for BRPF1 bromodomain, 3600 and 2310 nM for BRPF2 bromodomain, and >10 μM for full-length BRD4. In vitro proliferation assays performed with these compounds showed single digit micromolar inhibition for the ovarian cancer ES-2, small cell lung cancer DMS-53 and acute myeloid leukemia THP-1 cell lines. However, no anti-proliferative activity was observed in a number of other tumor cell lines derived from a variety of hematological and solid tumors. Altogether, these data show that inhibiting BRPF1 bromodomain has only a limited anti-proliferative effect on tumor cell lines, suggesting that other regions than the bromodomain of the protein account for the anti-proliferative effects seen in knock-down experiments. Citation Format: Bernard Haendler, Léa Bouché, Stephan Siegel, Amaury E. Fernandez-Montalvan, Tatsuo Sugawara, Julia Meier, Stefan Knapp, Vicki Gamble. Identification and characterization of BRPF1 bromodomain inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4691.

Colon cancer chemopreventive effects of baicalein, an active enteric microbiome metabolite from baicalin.

Baicalin is a major constituent of Scutellaria baicalensis, which is a commonly used herbal medicine in many Asian countries. After oral ingestion, intestinal micro-biota metabolism may change parent compound's structure and its biological activities. However, whether baicalin can be metabolized by enteric microbiota and the related anti-cancer activity is not clear. In this study, using human enteric microbiome incubation and HPLC analysis, we observed that baicalin can be quickly converted to baicalein. We compared the antiproliferative effects of baicalin and baicalein using a panel of human cancer cell lines, including three human colorectal cancer (CRC) cell lines. In vitro antiproliferative effects on CRC cells were verified using an in vivo xenograft nude mouse model. Baicalin showed limited antiproliferative effects on some of these cancer cell lines. Baicalein, however, showed significant antiproliferative effects in all the tested cancer cell lines, especially on HCT-116 human colorectal cancer cells. In vivo antitumor results supported our in vitro data. We demonstrated that baicalein exerts potent S phase cell cycle arrest and pro-apoptotic effects in HCT-116 cells. Baicalein induced the activation of caspase 3 and 9. The in silico modeling suggested that baicalein forms hydrogen bonds with residues Ser251 and Asp253 at the active site of caspase 3, while interactions with residues Leu227 and Asp228 in caspase 9 through its hydroxyl groups. Data from this study suggested that baicalein is a potent anticancer metabolite derived from S. baicalensis. Enteric microbiota play a key role in the colon cancer chemoprevention of S. baicalensis.

Effects of TGF-beta signalling inhibition with galunisertib (LY2157299) in hepatocellular carcinoma models and in ex vivo whole tumor tissue samples from patients.

Galunisertib (LY2157299) is a selective ATP-mimetic inhibitor of TGF-β receptor (TβR)-I activation currently under clinical investigation in hepatocellular carcinoma (HCC) patients. Our study explored the effects of galunisertib in vitro in HCC cell lines and ex vivo on patient samples. Galunisertib was evaluated in HepG2, Hep3B, Huh7, JHH6 and SK-HEP1 cells as well as in SK-HEP1-derived cells tolerant to sorafenib (SK-Sora) and sunitinib (SK-Suni). Exogenous stimulation of all HCC cell lines with TGF-β yielded downstream activation of p-Smad2 and p-Smad3 that was potently inhibited with galunisertib treatment at micromolar concentrations. Despite limited antiproliferative effects, galunisertib yielded potent anti-invasive properties. Tumor slices from 13 patients with HCC surgically resected were exposed ex vivo to 1 µM and 10 µM galunisertib, 5 µM sorafenib or a combination of both drugs for 48 hours. Galunisertib but not sorafenib decreased p-Smad2/3 downstream TGF-β signaling. Immunohistochemistry analysis of galunisertib and sorafenib-exposed samples showed a significant decrease of the proliferative marker Ki67 and increase of the apoptotic marker caspase-3. In combination, galunisertib potentiated the effect of sorafenib efficiently by inhibiting proliferation and increasing apoptosis. Our data suggest that galunisertib may be active in patients with HCC and could potentiate the effects of sorafenib.

Abstract 3761: Efficacy of the ATP-competitive mTOR inhibitor AZD8055 in PTEN-wild type cancer cells

Abstract Although the PI3K/Akt/mTOR pathway is frequently hyperactivated in human cancers due to the loss of function of the PTEN tumor suppressor, a proportion of cancer cells still carries wild type PTEN and thus the PI3K/Akt/mTOR pathway in these cells may remain unaltered. While it has been widely accepted that these cancer cells are less susceptible to the allosteric mTOR inhibitor rapamycin, it is unclear whether this is also the case for an ATP-competitive mTOR inhibitor. To address this question we evaluated the effects of the mTOR kinase inhibitor AZD8055 in a panel of wild type PTEN expressing cancer cells (6 cell lines of 4 tumor types). The phosphorylated Akt (S473) level was undetectable in most of these cells, suggesting the regular mTOR complex 1 (mTORC1) pathway and low mTOR complex 2 (mTORC2) activity. Cell proliferation was determined by WST-1 (MTT) assay and clonogenic survival was tested by colony formation assay. AZD8055 suppressed cell proliferation at the nanomolar level in a dose dependent manner, with IC50 values in a range of 6∼35 nM for different cell types. In contrast, rapamycin exhibited limited anti-proliferative effect, with less than 50% of inhibition observed even with higher doses in most cell lines. Similarly, clonogenic capacity was impaired by AZD8055 to a greater extent than was the case for rapamycin. To detect the mTOR pathway alterations by drug treatment, two cell lines with different expression levels of mTORC1 downstream pathways were selected: murine mammary tumor 4T1 cells (low phosphorylated S6 and high phosphorylated 4E-BP1) and human breast cancer MDA-MB-231 cells (normal phosphorylated S6 and moderate phosphorylated 4E-BP1). While the phospho-S6K (T389) and phospho-S6 (S235/236) pathway was potently inhibited by both agents, AZD8055 displayed more striking inhibition of the hierarchical 4E-BP1 phosphorylation (T37/46, T70 and S65) than rapamycin in both cells. In addition, both rapamycin and AZD8055 slightly increased Akt phosphorylation (T308 or S473), suggesting that the activation of the feedback loop controlled by S6K was functional. However, the PI3K inhibitor wortmannin did not sensitize cells to either agent, implying such feedback-dependent activation is dispensable for attenuating the drug response. Taken together, these findings indicate that AZD8055 significantly inhibits growth and survival of cancer cells that have wild type PTEN and normal PI3K/Akt/mTOR signaling, an observation mainly associated with the comprehensive activation of 4E-BP1. These data provide in vitro evidence in support of broadening the application of ATP-competitive mTOR inhibitors in cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3761. doi:1538-7445.AM2012-3761

Shortened interval of long-acting octreotide administration is effective in patients with well-differentiated neuroendocrine carcinomas in progression on standard doses.

In patients with well-differentiated (WD) neuroendocrine tumors (NET), long-acting octreotide (LAR), conventionally administered at a dose of 30 mg every 28 days, has well-documented anti-secretive but limited antiproliferative effects. The objective of this study was to evaluate a different schedule of LAR treatment consistent with a shorter interval between administrations (21 days) in WDNET patients with progressive disease at standard-dose interval. Twenty-eight patients followed for diagnosis and therapy of WDNET who had tumor progression during therapy with LAR 30 mg every 28 days were enrolled. Clinical, biological, and objective tumor response was evaluated after LAR 30 mg every 21 days. Time to progression was also evaluated after LAR 30 mg every 21 days and compared to LAR 30 mg every 28 days. The treatment with LAR 30 mg every 21 days resulted in complete and partial control of clinical symptoms in 40% and 60% of cases, respectively. Circulating neuroendocrine markers were significantly decreased in 30% of cases. A stabilization of disease was obtained in 93% and objective response in 7%. The median time to progression was significantly longer by using the shortened interval of LAR administration as compared to the standard one (30 vs 9 months, p<0.0001). The treatment was safe and well tolerated. The shortened schedule of LAR administration was able to re-institute control of clinical symptoms, to decrease level of circulating neuroendocrine markers and to increase time to progression in patients previously escaping from a standard schedule treatment.

TGF-β-induced growth inhibition in B-cell lymphoma correlates with Smad1/5 signalling and constitutively active p38 MAPK

BackgroundCytokines of the transforming growth factor β (TGF-β) superfamily exert effects on proliferation, apoptosis and differentiation in various cell types. Cancer cells frequently acquire resistance to the anti-proliferative signals of TGF-β, which can be due to mutations in proteins of the signalling cascade. We compared the TGF-β-related signalling properties in B-cell lymphoma cell lines that were sensitive or resistant to TGF-β-induced anti-proliferative effects.ResultsTGF-β sensitive cell lines expressed higher cell surface levels of the activin receptor-like kinase 5 (Alk-5), a TGF-β receptor type 1. The expression levels of the other TGF-β and bone morphogenetic protein receptors were comparable in the different cell lines. TGF-β-induced phosphorylation of Smad2 was similar in TGF-β sensitive and resistant cell lines. In contrast, activation of Smad1/5 was restricted to cells that were sensitive to growth inhibition by TGF-β. Moreover, with activin A we detected limited anti-proliferative effects, strong phosphorylation of Smad2, but no Smad1/5 phosphorylation. Up-regulation of the TGF-β target genes Id1 and Pai-1 was identified in the TGF-β sensitive cell lines. Constitutive phosphorylation of MAPK p38 was restricted to the TGF-β sensitive cell lines. Inhibition of p38 MAPK led to reduced sensitivity to TGF-β.ConclusionsWe suggest that phosphorylation of Smad1/5 is important for the anti-proliferative effects of TGF-β in B-cell lymphoma. Alk-5 was highly expressed in the sensitive cell lines, and might be important for signalling through Smad1/5. Our results indicate a role for p38 MAPK in the regulation of TGF-β-induced anti-proliferative effects.

Down-regulation of Polo-like Kinase 1 Elevates Drug Sensitivity of Breast Cancer CellsIn vitroandIn vivo

Human polo-like kinase 1 (Plk1) is a key player in different stages of mitosis and modulates the spindle checkpoint at the metaphase-anaphase transition. Overexpression of Plk1 is observed in various human tumors and it is a negative prognostic factor in patients suffering from diverse cancers. We used phosphorothioate antisense oligonucleotides (ASO) targeted against Plk1, together with paclitaxel, carboplatin, and Herceptin, for the treatment of breast cancer cells to identify conditions for enhanced drug sensitivity. After transfection of the breast cancer cell lines BT-474, MCF-7, and MDA-MB-435 with Plk1-specific ASOs, paclitaxel, carboplatin, or Herceptin was added and cell proliferation, cell cycle distribution, and apoptosis were measured. Whereas the dual treatment of breast cancer cells with Plk1-specific ASOs with carboplatin or Herceptin caused only a limited antiproliferative effect in breast cancer cells, we observed synergistic effects after combination of low doses of Plk1-specific ASOs with paclitaxel, which is used in a variety of clinical anticancer regimens. Plk1-specific ASOs also acted synergistically with paclitaxel in the arrest of the cell cycle at the G(2)-M phase and in the induction of apoptosis. Interestingly, in a human xenograft experiment using MDA-MB-435 cells, the combination of Plk1 ASOs with paclitaxel led to synergistic reduction of tumor growth after 3 weeks of treatment compared with either agent alone. This study suggests that antisense inhibitors against Plk1 at well-tolerated doses may be considered as highly efficient promoters for the antineoplastic potential of taxanes, such as paclitaxel, causing synergistic effects in breast cancer cells.

Possible involvement of m-calpain in vascular smooth muscle cell proliferation.

Vascular smooth muscle cell (VSMC) proliferation still remains a poorly understood process, although it is believed to play a critical role in pathological states, including atherosclerosis and hypertension. Several reports have suggested that proteases may be directly involved in this process; however, it was still unclear which protease is responsible for VSMC proliferation. In this study, by use of a cell-permeable calpain inhibitor (calpeptin; benzyloxycarbonyl-Leu-nLeu-H), its analogue (benzyloxycarbonyl-Leu-Met-H), the cell-impermeable serine protease inhibitor leupeptin, and antisense oligonucleotide against m-calpain to inhibit proliferation of primarily cultured human VSMCs, we investigated whether calcium-activated neutral protease (calpain) is involved in VSMC proliferation. Calpeptin and its analogue, more specific for m-calpain, equally inhibited the proliferation of VSMCs in a dose-related manner, whereas a more limited antiproliferative effect was observed in leupeptin-treated VSMCs. Antisense oligonucleotide against m-calpain, but not scrambled antisense, dose-dependently inhibited m-calpain expression and proliferation of VSMCs. Maximal inhibition was an approximately 50% reduction of cell number and m-calpain antigen observed at 50 micromol/L of antisense oligonucleotide. Calpeptin or antisense oligonucleotide against m-calpain increased the expression of the endogenous calpain substrate pp125FAK (focal adhesion kinase), whereas the expression of the endogenous calpain inhibitor calpastatin was not affected. These results suggest that the proliferation of VSMCs requires protease activity, some of which is due to m-calpain.