Decreased Expression Of p-ERK Research Articles

Abstract 5264: KRASG12C inhibition synergizes with checkpoint blockade in KRASG12C-mutated tumor model

Abstract Introduction: The KRASG12C mutation is found in 14% of non-small cell lung cancer (NSCLC) and 3% of colorectal cancer (CRC). Sotorasib (AMG510), which covalently binds to the mutated cysteine 12 of KRASG12C protein, became the first KRASG12C inhibitors approved by the FDA for KRASG12C-mutated NSCLC treatment. AMG510 not only increased apoptosis in cancer cells harboring KRASG12C mutation, but also re-modelled tumor microenvironment. Thus, to evaluate novel therapeutic strategies for treating KRASG12C-mutated tumors, we established a CT26-KRASG12C tumor model and explored the combination of AMG510 with anti-PD1 therapy. Methods: We knocked in KRASG12C mutation in murine CT26 colorectal cancer cells using CRISPR/Cas9 to generate CT26-KRASG12C cells. The mutation was confirmed by PCR sequencing. In vitro, CT26-KRASG12C cells were treated with AMG510 for 2h or 72h, and then p-ERK and cell viability were analyzed. In vivo, 3×105 CT26-KRASG12C cells were subcutaneously injected into BALB/c mice, and the tumor bearing mice were treated with AMG510 (30 mg/kg or 100 mg/kg, QD×21), anti-PD-1 (10 mg/kg, BIW×3) and combination of AMG510 and anti-PD-1. On day 5 after randomization, the composition of immune cells, including CD4+ T cells, CD8+ T cells, dendritic cells (DC), and natural killer (NK) cells in tumors were analyzed by flow cytometry. The mice with complete tumor regression were re-challenged with bilateral tumors of CT26-KRASG12C (3×105) and unrelated mouse breast tumor cells, 4T1 (3×105) to evaluate the effect on tumor growth. Results: Following confirmation of KRASG12C mutation, AMG510 treatment decreased p-ERK expression by ~50% and impaired the cell growth with IC50 value of 0.1802 μM in CT26-KRASG12C cells. In vivo, single treatment of AMG510 at 30 mg/kg or 100 mg/kg significantly inhibited tumor growth with TGI of 60% or 99% on day 15 after randomization, respectively (both p<0.001). Combined treatment of AMG510 (30 mg/kg or 100 mg/kg) and anti-PD-1 also led to significant tumor inhibition with TGI of 64% or 99% on the same day, respectively (both p<0.001), although PD-1 blockade didn’t show anti-tumor effect alone. Noticeably, combined treatment of AMG510 (100 mg/kg) and anti-PD-1 induced complete tumor regression in 3 of 10 mice, which remained cured 124 days later. Moreover, the combined treatment significantly improved survival compared to the AMG510 treatment alone. Notably, all 4T1 tumors grew, but none of the CT26-KRASG12C tumors were palpable in the cured mice after the re-challenge. In the control group, all tumors were well established. Additionally, combined treatment of AMG510 (100mg/kg) and anti-PD-1 markedly increased the infiltration of CD4+ T cells, DC, and NK cells in the tumors. Conclusion: The successfully established CT26-KRASG12C model is a useful tool for investigating therapeutic strategies to combine KRASG12C inhibitors with immunotherapy. Citation Format: Li Hua, Shuang Li, Chenpan Nie, Chenfei Zhang, Ludovic Bourre, Jingjing Wang. KRASG12C inhibition synergizes with checkpoint blockade in KRASG12C-mutated tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5264.

Combined Lycium barbarum Polysaccharides with Plasmon-Activated Water Affect IFN-γ/TNF-α Induced Inflammation in Caco-2 Cells.

The effects of Lycium barbarum polysaccharides (LBP) and plasmon-activated water (PAW) against IFN-γ/TNF-α induced inflammation in human colon Caco-2 cells were investigated. Cells were divided into the control, induction, LBP treatment (100-500 μg/mL), and combination groups with PAW. Inflammation was induced 24 h with 10 ng/mL IFN-γ when cell confluency reached >90%, and various doses of LBP with or without PAW were treated for 3 h, and subsequently 50 ng/mL TNF-α was added for another 24 h to provoke inflammation. Combination of LBP with PAW significantly decreased the secretion of IL-6 and IL-8. Cyclooxygenase-2 and inducible NO synthase expression was attenuated in all LBP-treated groups with or without PAW. NLRP3 inflammasome and related protein PYCARD expression were inhibited by LBP at the highest dose (500 μg/mL). All doses of LBP alone significantly decreased p-ERK expression, but combination with PAW increased p-ERK expression compared to those without PAW. Additionally, 250 and 500 μg/mL of LBP with or without PAW inhibited procaspase-3/caspase-3 expression. Therefore, LBP possesses anti-inflammation and anti-apoptosis by inhibiting the secretion of inflammatory cytokines and the expression of NLRP3 inflammasome-related protein. The combination with PAW exerts additive or synergistic effect on anti-inflammation.

Silencing of purinergic receptor P2Y2 inhibited enteric neural crest cell proliferation, invasion and migration via suppressing ERK signaling pathway in Hirschsprung disease.

The current study aimed to explore the effect and underlying mechanism of the purinergic receptor P2Y2 in regulating the loss of intestinal neurons and the intestinal neural crest in Hirschsprung's disease (HSCR). Western blotting was used to assess the expression levels of P2Y2 in colon tissues. An in vivo HSCR mouse model was established following treatment with benzalkonium chloride (BAC). We overexpressed or silenced P2Y2 in SH-SY5Y cells, and cell proliferation, migration, and invasion were subsequently investigated by CCK-8, wound healing, and transwell assays, respectively. Additionally, we implemented a xenograft model to assess the impact of P2Y2 on tumor growth as well as the expression of extracellular signal-regulated kinase (ERK). The results showed that the expression of P2Y2 protein in the colon tissues of patients with HSCR was lower than that in the normal colon tissues. P2Y2 expression is downregulated in the colon tissues of mice with HSCR. Additionally, P2Y2 silencing inhibited SH-SY5Y cell proliferation, invasion, and migration. Furthermore, adenosine 5'-triphosphate (ATP, a strong agonist of P2Y2)-induced P2Y2 overexpression enhanced the proliferation, invasion, and migration of SH-SY5Y cells. Immunofluorescence staining and western blot analysis revealed that P2Y2 silencing downregulated phosphorylated (p)-ERK in SH-SY5Y cells. In addition, treatment with PD98059, a p-ERK inhibitor, reversed the effects of ATP on SH-SY5Y cell proliferation, invasion, and migration. Finally, we demonstrated that P2Y2 silencing suppressed tumor growth and decreased p-ERK expression. Overall, the results of the present study suggest that P2Y2 plays an important role in HSCR pathogenesis. P2Y2 silencing inhibited the proliferation, invasion, and migration of nerve cells by suppressing the ERK signaling pathway. P2Y2 silencing could be considered an innovative and possible target for treating HSCR.

Functional imaging of RAS pathway targeting in malignant peripheral nerve sheath tumor cells and xenografts.

Malignant peripheral nerve sheath tumor (MPNST) is an aggressive form of soft-tissue sarcoma (STS) in children. Despite intensive therapy, relatively few children with metastatic and unresectable disease survive beyond three years. RAS pathway activation is common in MPNST, suggesting MEK pathway inhibition as a targeted therapy, but the impact on clinical outcome has been small to date. We conducted preclinical pharmacokinetic (PK) and pharmacodynamic studies of two MEK inhibitors, trametinib and selumetinib, in two MPNST models and analyzed tumors for intratumor drug levels. We then investigated 3'-deoxy-3'-[18 F]fluorothymidine (18 F-FLT) PET imaging followed by 18 F-FDG PET/CT imaging of MPNST xenografts coupled to short-term or longer-term treatment with selumetinib focusing on PET-based imaging as a biomarker of MEK inhibition. Trametinib decreased pERK expression in MPNST xenografts but did not prolong survival or decrease Ki67 expression. In contrast, selumetinib prolonged survival of animals bearing MPNST xenografts, and this correlated with decreased pERK and Ki67 staining. PK studies revealed a significantly higher fraction of unbound selumetinib within a responsive MPNST xenograft model. Thymidine uptake, assessed by 18 F-FLT PET/CT, positively correlated with Ki67 expression in different xenograft models and in response to selumetinib. The ability of MEK inhibitors to control MPNST growth cannot simply be predicted by serum drug levels or drug-induced changes in pERK expression. Tumor cell proliferation assessed by 18 F-FLT PET imaging might be useful as an early response marker to targeted therapies, including MEK inhibition, where a primary effect is cell-cycle arrest.

Protection of diabetes-induced kidney injury by phosphocreatine via the regulation of ERK/Nrf2/HO-1 signaling pathway

Diabetic nephropathy is the most common long-term complication of diabetes mellitus. The Methylglyoxal (MGO) production is mainly by metabolic pathways, such as lipolysis and glycolysis, its increases in the DM enhances oxidative stress and plays a crucial role in the diabetic nephrotic pathogenesis. Phosphocreatine (PCr) can improve lipopolysaccharide, ox-LDL-induced atherosclerosis, and alleviate vascular endothelial cell injury in diabetes. The aim of our present study is to examine the potential role of phosphocreatine (PCr) as a molecule protects against diabetes-induced Kidney Injury in-vitro and in-vivo through ERK/Nrf2/HO-1 signaling pathway. NRK-52E cells treatment with PCr obviously suppressed MGO-induced change of viability, apoptosis, coupled with decreased Bax/Bcl-2ratio, casapse-9 and caspase-3expressions. We determined the generation of reactive oxygen species (ROS) using membrane permeable fluorescent probe DCFH-DA as well as intracellular calcium by flow cytometry. ERK, Nrf2 and HO-1 expressions were determined by Western blot. PCr pretreatment significantly returned the oxidative stress enzymes to normal condition in-vitro and in-vivo. PCr pretreatment significantly reduced apoptosis, calcium and ROS production, induced by MGO, in NRK-52E cells. Moreover, pretreatment with PCr significantly inhibited cleaved caspase-3, cleaved caspase-9 and p-ERK expressions, while increased Nrf-2 and HO-1 expressions. Furthermore, PCr pretreatment significantly decreased p-ERK expression of MGO-induced injury in NRK-52E cells transfected with p-ERK cDNA. In conclusion, the renal protective effect of PCr in-vitro and in-vivo depends on suppressing apoptosis and ROS generation through ERK mediated Nrf-2/HO-1 pathway, suggesting that PCr may be a novel therapeutic candidate for the diabetic nephropathy treatment.

Abstract 1544: Cell penetrating single domain antibody (sdAb) SBT-100 binds KRAS and inhibits growth of human cancers with KRAS activating mutations

Abstract Background: Despite nearly fourth years of research, scientists have failed to develop a clinically viable therapy against KRAS, one of the deadliest families of cancer-causing proteins. Mutations in KRAS are prevalent amongst the top three most deadly cancer types in the United States: pancreatic (95%), colorectal (45%), and lung (35%). KRAS has been thought to be undruggable due to: 1) its intracellular location and lack of binding pockets for small molecules; 2) the high (pM) affinity of RAS for GTP precludes direct targeting of the nucleotide binding pocket; 3) high intracellular concentrations of GTP (uM) inhibits competition for the nucleotide-binding pocket by small molecules; and 4) possible toxicity. Mutations of KRAS result in it being perpetually turned on to propagate signal down the MAPK pathway. This results in constant production of P-ERK and plays an important role in malignant development. To overcome these challenges, Singh Biotechnology has developed SBT-100 a first in class & best in class novel sdAb that penetrates the cell membrane to bind KRAS to inhibit its GTPase activity. Methods: Human cancer cell lines were purchased from ATCC. BIAcore affinity studies were conducted by Precision Antibody. KRAS GTPase assay was purchased from Promega. Levels of P-ERK were determined using western blots. In vitro cell growth suppression was tested with MTT assay. Athymic nude mice for xenograft studies were purchased from Envigo. Results: SBT-100 binds KRAS with KD=10-9 and KRAS(G12D) with KD=10-7 as demonstrated by BIAcore affinity assay. Both SBT-100 and SBT-102 significantly inhibit KRAS GTPase activity in vitro and inhibition is comparable to polyclonal antibody to KRAS. Growth of MDA-MB-231 cells with KRAS(G13D) mutation and PANC-1 cells with KRAS(G12D) mutation are significantly decreased in the MTT assay when incubated with SBT-100. Additionally, same cell lines have significantly decreased P-ERK expression when cultured with SBT-100. Xenograft studies demonstrate significant growth suppression of MDA-MB-231 and PANC-1 when treated with SBT-100 in vivo. Conclusion: SBT-100, crosses the cell membrane, binds to KRAS intracellularly and its most common mutant with nanomolar affinity, inhibits KRAS GTPase activity, downregulates P-ERK signaling, and suppresses the growth of cancers cells in vitro and in vivo without showing any toxic effects. Citation Format: Sunanda Singh, Genoveva Murillo, Avani Singh, Samara Singh, Meenakshi Parihar, Anjali Singh, Rajendra Mehta, Ashutosh Parihar. Cell penetrating single domain antibody (sdAb) SBT-100 binds KRAS and inhibits growth of human cancers with KRAS activating mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1544.

Puerarin alleviate radicular pain from lumbar disc herniation by inhibiting ERK-dependent spinal microglia activation

Lumbar disc herniation is a common cause of radicular pain, but the mechanism remains ambiguous and the treatment stays unsatisfied. Many studies revealed a traditional Chinese medicine puerarin may moderate chronic pain from diabetes and nerve injury. Thus far, the role and mechanism of puerarin in radicular pain is still unknown. In this study, by using a rat model of lumbar disc herniation, which was induced by autologous nucleus pulposus (NP) implantation, the analgesic effect of puerarin on radicular pain was tested. Puerarin was delivered intraperitoneally form 1 h before surgery, and once daily for 7 days. The results demonstrated that NP implantation induced long-lasting pain, characterized by decrease of paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) in ipsilateral hindpaws, as long as day 20 after surgery. Spinal phosphorylated extracellular signal-regulated kinase (p-ERK) was up-regulated from day 5 to day 20 after surgery in ipsilateral but not contralateral side, and p-ERK was mainly co-localized with microglia. Puerarin decreased p-ERK expression from day 7 to day 20 after surgery. Puerarin or ERK inhibitor PD98059 alleviated pain behaviors, decreased expression of microglia marker ionized calcium-binding adaptor molecule 1 (Iba-1) in rats with NP implantation. The results suggested puerarin may alleviate radicular pain by inhibiting ERK-dependent or accompanied spinal microglia activation.

Preventative effects of resveratrol and estradiol on streptozotocin-induced diabetes in ovariectomized mice and the related mechanisms.

Resveratrol, a non-flavonoid polyphenolic compound, is structurally and functionally similar to estrogen and has drawn great attention for its potentially beneficial effects on diabetes. However, it is not known whether it shares the same protective effect against diabetes as estrogen and the underlying mechanisms. The aim of the present study was to investigate the protective effects of phytoestrogen resveratrol and exogenous 17β-estradiol against streptozotocin (STZ)-induced type 1 diabetes. Female mice were ovariectomized (OVX) and chronically injected with different concentrations of resveratrol (0.1, 1 or 10 mg/kg) and 17β-estradiol (0.01, 0.1 or 1 mg/kg) subcutaneously for 4 weeks, and the levels of blood glucose, plasma insulin, plasma antioxidant capacity, the changes of pancreatic islet cells and the expressions of glucose transporter 4 (GLUT4), insulin receptor substrate 1 (IRS-1) and phosphorylation of extracellular signal-regulated kinase (p-ERK) were detected. Resveratrol and 17β-estradiol significantly inhibited the increase of the blood glucose level and the rise of plasma malondialdehyde in STZ-induced diabetic mice, improved the levels of plasma antioxidant capacity and plasma insulin, protected the pancreatic islet cells, and increased the expressions of GLUT4 and IRS-1, but decreased p-ERK expression in skeletal muscle and myocardial tissue. The results suggest that resveratrol or 17β-estradiol shows obvious protection against STZ-induced diabetes in OVX mice, the mechanisms probably involve their ameliorating antioxidant activities and islet function, promoting muscle glucose uptake and inhibiting the expression of p-ERK.

Leukocyte Specific Protein‐1 (LSP1) Functions as a Suppressor of Proliferation During Liver Regeneration After Partial Hepatectomy (PHx) and in Primary Hepatocytes

Leukocyte specific protein‐1 (LSP1), an f‐actin binding protein expressed in hematopoietic cells and hepatocytes, is the gene with the highest copy number variation in human hepatocellular carcinoma. Our previous work has demonstrated that loss of LSP1 expression leads to increased proliferation and migration of rat hepatocellular carcinoma cells. We have also demonstrated that overexpression of LSP1 in mouse liver through hydrodynamic tail vein injection of LSP1 plasmid DNA results in a significant decrease in proliferation on day 2 after partial hepatectomy (PHx). To further characterize the role of LSP1 in liver regeneration, we performed 2/3 PHx on both a global LSP1 knockout mouse as well as a hepatocyte specific LSP1 transgenic mouse model and analyzed proliferation using ki67 immunohistochemistry as well as western blot analysis of ERK signaling. Since expression of LSP1 leads to decreased proliferation, we hypothesized that the knockout mouse model would exhibit increased proliferation and activation of ERK whereas the transgenic mouse model would display decreased proliferation and pERK expression. LSP1 knockout mice displayed an increase in Ki67 labeling on day 4 after PHx and western blot analysis shows increased pERK expression after PHx in comparison to controls. In the transgenic mouse model, a significant decrease in Ki67 positive hepatocytes was observed on day 4 after PHx as well as decreased pERK expression. Transgenic mice also displayed decreased liver to body weight ratios on day 7 following PHx. Hepatocytes isolated from knockout mice exhibited increased proliferation in the absence of growth factors compared to controls whereas transgenic mouse hepatocytes displayed decreased BrdU incorporation in both the presence and absence of growth factors. LSP1 knockout hepatocytes and transgenic hepatocytes also displayed increased and decreased pERK expression, respectively. Our results demonstrate that LSP1 functions as an inhibitor of proliferation in primary hepatocytes as well as during liver regeneration following PHx. Future studies aim to address the signaling pathways downstream of LSP1 that effect proliferation during liver regeneration as well as how LSP1 functions in hepatocarcinogenesis mechanisms and sensitivity to sorafenib.Support or Funding InformationMenten Endowment, University of Pittsburgh

Abstract 1680: Targeting subcellular localization of Kaiso to limit dissemination through MAPK signaling

Abstract Breast cancer is the second most commonly diagnosed cancer among American women. The American Cancer Society has estimated over 230,000 new cases of breast cancer, and over 40,000 deaths for the year 2015. Unfortunately, metastasis is the major contributor for the significant number of these deaths. The Epithelial-Mesenchymal Transition (EMT) is promoted by the Master Regulator known as Kaiso, a transcriptional repressor that is a member of the BTB/POZ family of zinc finger transcription factors. We have previously demonstrated that Epidermal Growth Factor Receptor (EGFR) signaling shuttles Kaiso into the nucleus, although, its mechanism is still not understood. EGFR signaling activates multiple downstream signaling cascades including the MAP Kinase cascade, which has a significant influence on proliferation, cell adhesion, gene expression, and cell survival. Mesenchymal cells demonstrate high levels of MAPK activity and disruption or inhibition of the cascade has proven to be a method for decreasing tumor growth. Therefore we hypothesize that subcellular localization and expression of Kaiso is modulated by EGFR induced MAPK activity. Using the TCGA Breast cancer dataset, we observed that Kaiso was altered in about 11% of patients, compared to 15% of E-cad expression and 13% of MAPK3 of 959 patients/cases of Breast cancers. MAPK3 (eRK1); MAPK6, MAPK7, MAPK9 AND MAPK14 (p38) seems to have trends in mutual exclusivity or co-occurrence with Kaiso (ZBTB33). To determine if MAPK3 is responsible for increased Kaiso expression, MCF-7 cells, which express low levels of Kaiso in the cytoplasm, were treated with 10ng/ml of EGF in the presence or absence of MAPK inhibitor, 20μM PD98093. EGF stimulation caused robust increase in Kaiso protein expression, while PD98093 inhibited EGF induced expression. Similarly, in Kaiso overexpressing, MDA-MB-231, blocking EGFR kinase activity with 500nM PD153035 resulted in an opposite effect as EGF stimulation in MCF-7 cells. Furthermore, we observed a significant decrease in p-ERK expression, re-expression of E-cadherin, and low EGFR expression levels in sh-Kaiso MDA-MB-231 cells compared to sh-Scr cells. The decreased p-ERK expression in sh-Kaiso MDA-MB-231 cells was also associated with decreased expression of mesenchymal markers ZEB1, ZEB2, Twist and SNAIL at both the mRNA and protein levels compared to the scrambled control. Similarly, we observed significant decrease in the somatic cell reprogramming factors Klf4, Oct4, Nanog expression in sh-Kaiso MDA-MB-231 as well, which coincide with a reversal of EMT. To our knowledge, this is the first study to investigate the molecular mechanism and/or functional consequences of increased Kaiso expression in breast cancer, and highlights the multiple roles that Kaiso plays in promoting aggressive disease. Citation Format: Shweta Tripathi, Balasubramanyam Karanam, Bo Ma, Alan Wells, Clayton Yates. Targeting subcellular localization of Kaiso to limit dissemination through MAPK signaling. [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 1680.

Acid-sensing ion channel 3 decreases phosphorylation of extracellular signal-regulated kinases and induces synoviocyte cell death by increasing intracellular calcium.

IntroductionAcid-sensing ion channel 3 (ASIC3) is expressed in synoviocytes, activated by decreases in pH, and reduces inflammation in animal models of inflammatory arthritis. The purpose of the current study was to characterize potential mechanisms underlying the control of inflammation by ASIC3 in fibroblast-like synoviocytes (FLS).MethodsExperiments were performed in cultured FLS from wild-type (WT) and ASIC3-/- mice, ASIC1-/- mice, and people with rheumatoid arthritis. We assessed the effects of acidic pH with and without interleukin-1β on FLS and the role of ASICs in modulating intracellular calcium [Ca2+]i, mitogen activated kinase (MAP kinase) expression, and cell death. [Ca2+]i was assessed by fluorescent calcium imaging, MAP kinases were measured by Western Blots; ASIC, cytokine and protease mRNA expression were measured by quantitative PCR and cell death was measured with a LIVE/DEAD assay.ResultsAcidic pH increased [Ca2+]i and decreased p-ERK expression in WT FLS; these effects were significantly smaller in ASIC3-/- FLS and were prevented by blockade of [Ca2+]i. Blockade of protein phosphatase 2A (PP2A) prevented the pH-induced decreases in p-ERK. In WT FLS, IL-1β increases ASIC3 mRNA, and when combined with acidic pH enhances [Ca2+]i, p-ERK, IL-6 and metalloprotienase mRNA, and cell death. Inhibitors of [Ca2+]i and ERK prevented cell death induced by pH 6.0 in combination with IL-1β in WT FLS.ConclusionsDecreased pH activates ASIC3 resulting in increased [Ca2+]i, and decreased p-ERK. Under inflammatory conditions, acidic pH results in enhanced [Ca2+]i and phosphorylation of extracellular signal-regulated kinase that leads to cell death. Thus, activation of ASIC3 on FLS by acidic pH from an inflamed joint could limit synovial proliferation resulting in reduced accumulation of inflammatory mediators and subsequent joint damage.

Regulation of Autophagy Via PERK-eIF2α Effectively Relieve the Radiation Myelitis Induced by Iodine-125

Radiation myelitis is the most serious complication in clinical radiotherapy for spinal metastases. We previously showed that 125I brachytherapy induced apoptosis of spinal cord neurons accompanied by autophagy. In this study, we further investigated the mechanism by which 125I radiation triggered autophagy in neural cells. We found that autophagy induced by 125I radiation was involved in endoplasmic reticulum (ER) stress and mainly dependent on PERK-eIF2α pathway. The expressions of LC3II, ATG12 and PI3K were significantly suppressed in PERK knockout neural cells. Meanwhile, the expressions of phosphorylated-Akt s473 and caspase3/8 all significantly increased in neural cells transfected with a PERK siRNA and which enhanced apoptosis of neurons after 125I radiation. The results were consistent with that by MTT and Annexin-FITC/PT staining. In annimal model of banna pigs with radiation myelitis caused by 125I brachytherapy, we have successfully decreased PERK expression by intrathecal administration of the lentivirus vector. The apoptosis rate was significantly higher than that in control group and which deteriorated radiation myelitis of banna pigs. Thus, autophagy caused by 125I radiation was mainly as an attempt of cell survival at an early stage, but it would be a self-destructive process and promoted the process of apoptosis and necrosis radiated by 125I for more than 72 hours. The study would be useful and helpful to maximize efficiency of radiation therapy in clinical therapy.

Abstract 3676: Selective modulation of CHOP/GADD153 in prostate cancer cells by alpha-mangostin promotes cell death.

Abstract Background: It is well-established that during endoplasmic reticulum (ER) stress cells utilize several cell signal pathways collectively named “unfolded protein reaction (UPR)” to re-establish homeostasis and promote cell survival. The upregulation of UPR proteins to an extent is thought to be a growth advantage for cancer cells. However, chronic ER stress or prolonged UPR will lead to apoptosis. Alpha-mangostin, a xanthone from mangosteen fruit, has been reported to effectively induce prostate cancer cell apoptosis in vitro and suppress xenograft tumor growth. Thus, we investigated the alteration of ER stress-related proteins in mangosteen fruit extract (MFE)- or α-Mangostin-treated prostate cancer cells as well as its correlation with apoptosis. Methods: Two human prostate cancer cell lines, 22Rv1 and LNCaP, and a prostate epithelial cell line were treated with MFE or α-mangostin. Flow cytometry, MTT and BrdU were used to evaluate cell apoptosis, viability and proliferation. Western blot and fluorescent microscopy were used to detect proteins involved in apoptosis or ER stress. We also evaluated the anti-cancer activity of MFE and α-mangostin in nude mice inoculated with 22Rv1 cells. In addition, pharmacokinetics data of α-mangostin was collected on wild type C57BL6 mice for toxicity concern. Results: MFE increased apoptosis, decreased viability and inhibited proliferation in prostate cancer cell lines. Both MFE and α-mangostin significantly upregulated the expression of ER stress proteins, such as PERK, CHOP and IRE1α. Both MFE and α-mangostin also effectively suppressed tumor growth in a xenograft tumor model without affecting body weight. Interestingly, CHOP knockdown enhanced apoptosis in α-mangostin-treated prostate cancer cells; both MFE and α-mangostin decreased PERK expression and did not induce ER stress in prostate epithelial cells (PrECs) from prostate cancer patients. Conclusions: Both MFE and α-mangostin selectively promote ER stress in prostate cancer cells and inhibit prostate cancer cell growth in a xenograft mouse model without observable toxicity, suggesting further research is needed to evaluate its potential as an anti-cancer agent. Citation Format: Gongbo Li, Sakina Petiwala, Dana Pierce, Jeremy Johnson. Selective modulation of CHOP/GADD153 in prostate cancer cells by alpha-mangostin promotes cell death. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3676. doi:10.1158/1538-7445.AM2013-3676

N-3 Fatty acids block TNF-α-stimulated MCP-1 expression in rat mesangial cells

Monocyte chemoattractant protein 1 (MCP-1) is a CC cytokine that fundamentally contributes to the pathogenesis of inflammatory renal disease. MCP-1 is highly expressed in cytokine-stimulated mesangial cells in vitro and following glomerular injury in vivo. Interventions to limit MCP-1 expression are commonly effective in assorted experimental models. Fish oil, an abundant source of n-3 fatty acids, has anti-inflammatory properties, the basis of which remains incompletely defined. We examined potential mechanisms whereby fish oil reduces MCP-1 expression and thereby suppresses inflammatory responses to tissue injury. Cultured mesangial cells were treated with TNF-α in the presence of the n-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA); equimolar concentrations of the n-6 fatty acids LA and OA served as controls. MCP-1 mRNA expression was assessed by Northern blotting, and transcriptional activity of the MCP-1 promoter was assessed by transient transfection. The involvement of the ERK and NF-κB pathways was evaluated through transfection analysis and the use of the MEK inhibitor U0126. DHA and EPA decreased TNF-α-stimulated MCP-1 mRNA expression by decreasing transcription of the MCP-1 gene. DHA and EPA decreased p-ERK expression and nuclear translocation of NF-κB, both of which are necessary for TNF-α-stimulated MCP-1 expression. Both NF-κB and AP-1 sites were involved in transcriptional regulation of the MCP-1 gene by DHA and EPA. We conclude that DHA and EPA inhibit TNF-α-stimulated transcription of the MCP-1 gene through interaction of signaling pathways involving ERK and NF-κB. We speculate that such effects may contribute to the salutary effect of fish oil in renal and vascular disease.

Effectively targeting BRAF in melanoma: a formidable challenge

Effectively targeting BRAF in melanoma: a formidable challenge