Tocilizumab Exerts Anti-Tumor Effects on Colorectal Carcinoma Cell Xenografts Corresponding to Expression Levels of Interleukin-6 Receptor.

The use of tocilizumab against the interleukin-6 receptor (IL-6R) has been demonstrated as inhibiting the progression of diverse cancers in vitro and in vivo. Nonetheless, evidence regarding the anti-tumor effects of tocilizumab on human colorectal carcinoma (CRC) corresponding to IL-6R expression levels remains scarce. To investigate the influence of IL-6R expression, SW480 and HT-29 cells inoculated subcutaneously into NU/NU mice were used as human CRC xenograft models with anti-IL-6R antibody (tocilizumab) therapy. The IL-6R expression levels, histology of CRC growth/invasiveness, and tumor growth-related signaling pathway were estimated by H&E and immunohistochemical staining. SW480 tumor cells with higher IL-6R expression levels showed better responsiveness in tocilizumab therapy than in the treated HT-29 group. Likewise, therapeutic effects of tocilizumab on the proliferative ability with mitotic index and Ki-67 expressions, invasiveness with MMP-9 proteinase expressions, and ERK 1/2 and STAT3 signaling transduction in the SW480 treatment group were superior to the HT-29 treatment group. In light of our results, IL-6R is the key indicator for the efficacy of tocilizumab treatment in CRC xenografts. From the perspective of precision medicine, tumor response to anti-IL-6R antibody therapy could be predicted on the basis of IL-6R expression levels. In this manner, tocilizumab may serve as a targeted and promising anti-CRC therapy.

Synergistic Chemopreventive Effects of a Novel Combined Plant Extract Comprising Gallic Acid and Hesperidin on Colorectal Cancer.

BACKGROUND/AIM: Colorectal cancer (CRC) is the third most common cancer with a high mortality rate worldwide. Although gallic acid and hesperidin exert anticancer activity, synergistic effects of gallic acid and hesperidin against CRC remain elusive. This study aims to investigate the therapeutic mechanism of a novel combination of gallic acid and hesperidin against CRC cell growth, including cell viability, cell-cycle-associated proteins, spheroid formation, and stemness. METHODS: Gallic acid and hesperidin derived from Hakka pomelo tea (HPT) were detected by colorimetric methods and high-performance liquid chromatography using ethyl acetate as an extraction medium. CRC cell lines (HT-29 and HCT-116) treated with the combined extract were investigated in our study for cell viability (trypan blue or soft agar colony formation assay), cell cycle (propidium iodide staining), cell-cycle-associated proteins (immunoblotting), and stem cell markers (immunohistochemistry staining). RESULTS: Compared with other extraction methods, HPT extraction using an ethyl acetate medium exerts the most potent effect on inhibiting HT-29 cell growth in a dose-dependent manner. Furthermore, the treatment with combined extract had a higher inhibitory effect on CRC cell viability than gallic acid or hesperidin alone. The underlying mechanism was involved in G1-phase arrest and Cip1/p21 upregulation that could attenuate HCT-116 cell proliferation (Ki-67), stemness (CD-133), and spheroid growth in a 3D formation assay mimicking in vivo tumorigenesis. CONCLUSION: Gallic acid and hesperidin exert synergistic effects on cell growth, spheroids, and stemness of CRC and may serve as a potential chemopreventive agent. Further testing for the safety and effectiveness of the combined extract in large-scale randomized trials is required.

A Fc-VEGF chimeric fusion enhances PD-L1 immunotherapy via inducing immune reprogramming and infiltration in the immunosuppressive tumor microenvironment.

BACKGROUND: Immunotherapy is an emerging cancer therapy with potential great success; however, immune checkpoint inhibitor (e.g., anti-PD-1) has response rates of only 10-30% in solid tumor because of the immunosuppressive tumor microenvironment (TME). This affliction can be solved by vascular normalization and TME reprogramming. METHODS: By using the single-cell RNA sequencing (scRNAseq) approach, we tried to find out the reprogramming mechanism that the Fc-VEGF chimeric antibody drug (Fc-VFD) enhances immune cell infiltration in the TME. RESULTS: In this work, we showed that Fc-VEGF121-VEGF165 (Fc-VEGF chimeric antibody drug, Fc-VFD) arrests excess angiogenesis and tumor growth through vascular normalization using in vitro and in vivo studies. The results confirmed that the treatment of Fc-VFD increases immune cell infiltration including cytotoxic T, NK, and M1-macrophages cells. Indeed, Fc-VFD inhibits Lon-induced M2 macrophages polarization that induces angiogenesis. Furthermore, Fc-VFD inhibits the secretion of VEGF-A, IL-6, TGF-ß, or IL-10 from endothelial, cancer cells, and M2 macrophage, which reprograms immunosuppressive TME. Importantly, Fc-VFD enhances the synergistic effect on the combination immunotherapy with anti-PD-L1 in vivo. CONCLUSIONS: In short, Fc-VFD fusion normalizes intratumor vasculature to reprogram the immunosuppressive TME and enhance cancer immunotherapy.

Translational Medicine in Uremic Vascular Calcification: Scavenging ROS Attenuates p-Cresyl Sulfate-Activated Caspase-1, NLRP3 Inflammasome and Eicosanoid Inflammation in Human Arterial Smooth Muscle Cells.

We formerly proved that uremic vascular calcification (UVC) correlates tightly with oxidative elastic lamina (EL) injury and two cell fates (apoptosis and osteocytic conversion) in smooth muscle cells (SMC) of chronic kidney disease (CKD) patients and eliminating p-cresyl sulfate (PCS)-activated intracellular ROS ameliorates the MAPK signaling pathway in a human arterial SMC (HASMC) model. Nonetheless, whether ROS scavenger attenuates PCS-triggered inflammasome activation and eicosanoid inflammation in the UVC process remains unknown. Patients with lower extremity amputation were categorized into CKD and normal control group according to renal function. We used immunohistochemistry stain to analyze UVC in arterial specimens, including oxidative injury (8-hydroxy-2'-deoxyguanosine (8-OHdG) and internal EL disruption), cytosolic phospholipase A2 (cPLA2), cyclooxygenase 2 (COX2), interleukin-1 beta (IL-1ß), caspase-1 and NLRP3. To simulate the patho-mechanism of human UVC, the therapeutic effects of ROS scavenger on PCS-triggered inflammatory pathways was explored in a HASMC model. We found CKD patients had higher circulating levels of PCS and an increase in medial arterial calcification than the control group. In CKD arteries, the severity of UVC corresponded with expressions of oxidative EL disruption and 8-OHdG. Furthermore, coupling expressions of cPLA2 and COX2 were accentuated in CKD arteries, indicative of eicosanoid inflammation. Notably, tissue expressions of IL-1ß, caspase-1 and NLRP3 were enhanced in parallel with UVC severity, indicative of inflammasome activation. From bedside to bench, ROS scavenger attenuates PCS-activated expressions of cPLA2/COX2, pro-caspase-1 and NLRP3 in the HASMC model. UVC as an inevitable outcome is predictive of death in CKD patients. Nonetheless, UVC remain pharmacoresistant despite the evolution of treatment for mineral-parathyroid hormone-vitamin D axis. Beyond the mineral dysregulation, the stimulation of pro-oxidant PCS alone results in eicosanoid inflammation and inflammasome activation. Concerning the key role of Caspase-1 in pyroptosis, cell fates of HASMC in uremic milieu are not limited to apoptosis and osteogenesis. In view of this, reducing ROS and PCS may act as a therapeutic strategy for UVC-related cardiovascular events in CKD patients.

Antibody to Interleukin-6 Receptor Inhibits In Vivo Growth of Human Colorectal Carcinoma Cell Xenografts.

BACKGROUND: Interleukin-6 receptor antibody (IL6R) inhibits colony formation and invasion by colorectal carcinoma (CRC) in vitro. We examined the effect of IL6R antibody on tumor growth of CRC xenografts in vivo. MATERIALS AND METHODS: SW480 cells inoculated subcutaneously into NU/NU mice were treated with anti-IL6R and tumor histology and growth-related signaling were subsequently estimated by hematoxylin and eosin and immunohistochemical staining. RESULTS: Tumor growth was inhibited by anti-IL6R treatment at dosages of both 0.1 and 1.0 mg/kg. Tumor cells had invaded into surrounding tissues in untreated mice, while there was no invasion of tumors in the IL6R antibody-treated mice. The expression of Ki-67, signal transducer and activator of transcription protein 3 (STAT3) and phosphor-extracellular signal-regulated kinase 1 and 2 (ERK1/2) were suppressed in anti-IL6R-treated tumors. CONCLUSION: IL6R antibody inhibited tumor growth and invasiveness in vivo by suppressing the expression of Ki-67, STAT3 and phosphor-ERK1/2. The results imply that the anti-IL6R may be a promising targeted drug for CRC.

Synergistic Effect of Combined Treatment with Longan Flower Extract and 5-Fluorouracil on Colorectal Cancer Cells.

To investigate the influence of longan flower extract (LFE) on the sensitization of colorectal cancer (CRC) cells to 5-fluorouracil (5-FU) treatment, HT-29, Colo 320DM and SW480 cells were treated with LFE and 5-FU alone and in combination, and the cell viability was then assessed by trypan blue exclusion, the cell cycle by propidium iodide staining, the mitochondria membrane potential by rhodamine 123 staining, and the expression levels of associated genes by immunoblotting and quantitative real-time polymerase chain reaction. LFE and 5-FU synergistically inhibited cell proliferation of HT-29 and Colo 320DM cells. Combined treatment also elevated the level of loss of mitochondria membrane potential of these two CRC cells and arrested HT-29 cells in the S phase of the cell cycle, in association with down-regulation of cyclin A mRNA expression. LFE synergistically potentiated chemosensitivity to 5-FU in at least two CRC cell lines. The results indicated that LFE has potential as a novel agent for the sensitization of CRC cells to 5-FU.

Longan flower proanthocyanidins induce apoptosis in HT-29 colorectal carcinoma spheroids.

AIM OF STUDY: Proanthocyanidin-rich longan flower extract (LFP) has been previously shown to inhibit the proliferation and anchorage-independent growth in soft agar of two colorectal carcinoma (CRC) cells in vitro. In this report, we further examined the effects of LFP in a CRC spheroid model. MATERIALS AND METHODS: A liquid-overlay assay employing HT-29 spheroids was used to evaluate the effects of LFP on cancer cell tumorigenesis, viability, and apoptosis. Associated effects on signaling path ways (epidermal growth factor receptor [EGFR], Akt) and apoptotic regulators were measured using Western blot. RESULTS: Treatment with LFP up to 200 µg/ml inhibited tumor growth in a dose-dependent manner and induced prominent apoptosis as measured by annexin V staining. Cells treated with LFP showed decreased EGFR and Akt phosphorylation with decreased expression of B-cell lymphoma 2. CONCLUSION: The ability of LFP to induce apoptosis in CRC spheroids warrants further investigation of its composition and identification of tumor-active components.

Litchi seed extract inhibits epidermal growth factor receptor signaling and growth of Two Non-small cell lung carcinoma cells.

BACKGROUND: Litchi seeds possess rich amounts of phenolics and have been shown to inhibit proliferation of several types of cancer cells. However, the suppression of EGFR signaling in non-small cell lung cancer (NSCLC) by litchi seed extract (LCSE) has not been fully understood. METHODS: In this study, the effects of LCSE on EGFR signaling, cell proliferation, the cell cycle and apoptosis in A549 adenocarcinoma cells and NCI- H661 large-cell carcinoma cells were examined. RESULTS: The results demonstrated that LCSE potently reduced the number of cancer cells and induced growth inhibition, cell-cycle arrest in the G1 or G2/M phase, and apoptotic death in the cellular experiment. Only low cytotoxicity effect was noted in normal lung MRC-5 cells. LCSE also suppressed cyclins and Bcl-2 and elevated Kip1/p27, Bax and caspase 8, 9 and 3 activities, which are closely associated with the downregulation of EGFR and its downstream Akt and Erk-1/-2 signaling. CONCLUSION: The results implied that LCSE suppressed EGFR signaling and inhibited NSCLC cell growth. This study provided in vitro evidence that LCSE could serve as a potential agent for the adjuvant treatment of NSCLC.

Rab11 collaborates E-cadherin to promote collective cell migration and indicates a poor prognosis in colorectal carcinoma.

BACKGROUND: Collective cell migration, whereby the cell-cell contacts such as E-cadherin are maintained during migration, has only recently emerged, and its detailed mechanisms are still unclear. In this study, the role of Rab11, which functions in recycling endosomes, and its relationship to E-cadherin in colorectal carcinoma were identified, and the role of Rab11 in the collective cell migration of colon cancer cells was clarified. MATERIALS AND METHODS: A total of 107 patients with surgically resected colorectal carcinoma were enrolled in this immunohistochemical study. Relationships between the overexpression of Rab11 and E-cadherin and survival were evaluated. The cell biology of Rab11 overexpression or knock-down in HT-29 colon cells was studied. RESULTS: The expression of Rab11 and E-cadherin was not correlated with the stage of cancer or lymph node metastasis. However, the overall survival was poor in the group of 67 patients with duo-positive Rab11 and E-cadherin expression compared to the group (40 patients) without dual-positive expression (P = 0·038). Rab11 was demonstrated to have a physical interaction with E-cadherin, and overexpression of Rab11 was found to promote collective cell migration through the increased distribution of E-cadherin, which enhanced cell-cell connections. In addition, Rac1 activation and matrix metalloproteinase-2 expressions were upregulated upon Rab11 expression. CONCLUSIONS: This study demonstrated that Rab11 and E-cadherin expressions are indicators of poor survival time in colorectal carcinoma, but that Rab11 overexpression may contribute to increased collective cell invasion in colorectal carcinoma.

Cost-Effective Hierarchical Catalysts for Promoting Hydrogen Release from Complex Hydrides.

Fe nanoparticles (∼10 nm), used to grow carbon nanotubes (CNTs), have an outstanding ability to catalyze the dehydrogenation of LiAlH4 . The CNTs help connect Fe and LiAlH4 and create microchannels among the composite, thus promoting the release of hydrogen. Inspired by these results, a supercritical-CO2 -fluid-assisted deposition technique is employed to decorate the Fe/CNTs with highly dispersed nanosized Ni (∼2 nm in diameter) for better performance. With the incorporation of 10 wt % of this hierarchical catalyst (Ni/Fe/CNTs), the initial dehydrogenation temperature of LiAlH4 is decreased from ∼135 to ∼40 °C. At 100 °C, this catalyzed LiAlH4 takes only ∼0.1 h to release 4.5 wt % hydrogen, which is more than 100 times faster than the time needed with pristine LiAlH4 . The dehydrogenation mechanism of the complex hydride is examined using in situ synchrotron X-ray diffraction.

Curcumin Inhibits Invasiveness and Epithelial-Mesenchymal Transition in Oral Squamous Cell Carcinoma Through Reducing Matrix Metalloproteinase 2, 9 and Modulating p53-E-Cadherin Pathway.

HYPOTHESES: Epithelial-mesenchymal transition (EMT) and invasion play a critical role in cancer progression and metastasis. We have shown that low E-cadherin and high Twist expression are significantly correlated with prognostic survival prediction in oral squamous cell carcinoma (OSCC). This study aimed to determine the anti-invasive effect of curcumin on the expression of matrix metalloproteinases (MMPs) and of EMT regulators in OSCC. METHODS: SCC-25 cells were treated with curcumin, and cell proliferation, invasion, and expression of MMPs and EMT regulators were assessed for cell viability by trypan blue exclusion, for invasion by Matrigel invasion chamber, and for EMT regulators and MMP changes in the levels of proteins by immunoblotting. RESULTS: Our data showed that curcumin treatment not only decreased the expression of MMP-2 and MMP-9 to inhibit invasiveness in oral cancer but also modulated the expression of EMT markers, such as Snail, Twist, and E-cadherin, and induced p53 expression that is crucial to EMT repression. CONCLUSION: Curcumin has the potential to become an adjunctive regimen for the prevention of cancer progression and metastasis in oral cancer.

Rab11 regulates E-cadherin expression and induces cell transformation in colorectal carcinoma.

BACKGROUND: In the process of epithelial mesenchymal transition EMT, the disassembly of junctional adhesion complexes such as E-cadherin is a remarkable sign during changes in cell morphology and polarity. However, E-cadherin expression is dynamic, and is regulated by the cellular endocytic system; it is also involved in cell signaling mechanisms. In this study, we investigated the role of E-cadherin in colorectal tumors and the relationship with recycling endosome protein Rab11 in colon cell transformation. METHODS: For tissue screening, the expressions of E-cadherin and Rab11 in colorectal tumors were identified by immunohistochemistry in 113 patients with colorectal carcinoma. For the in vitro cell experiment, GFP-tagged Rab11 plasmid was transfected into HT29 colon cells, E-cadherin expression and cell transformation were monitored by Western blot and confocal microscopy. RESULTS: In immunohistochemistry, the mean score of E-cadherin in tumor and normal tissues was 1.41 ± 0.06 and 1.08 ± 0.06 (p < 0.05). The mean score of Rab11 in tumor and normal tissues was 0.51 ± 0.05 and 0.18 ± 0.02 (p < 0.05). Synchronous overexpression of E-cadherin and Rab11 was noted in 74 patients (66.5%) with colorectal carcinoma. When GFP-tagged Rab11 plasmid was overexpressed in cultured colon cell line HT-29, the E-cadherin expression was up-regulated, and cell membrane protrusion was induced, which resulted in cell transformation and cell migration. CONCLUSIONS: This study demonstrated the importance of the overexpression of Rab11 and E-cadherin in colorectal cancer. The results indicated that Rab11 together with E-cadherin might be potential markers for colorectal cancer progression and treatment.

Buckyball-, carbon nanotube-, graphite-, and graphene-enhanced dehydrogenation of lithium aluminum hydride.

Compared to C60, carbon nanotubes, and graphite, graphene more effectively lowers the dehydrogenation temperature and improves the dehydrogenation kinetics of LiAlH4. With 15 wt% graphene incorporation, the initial hydrogen release temperature is ~80 °C (60 °C lower than that of pristine LiAlH4).

The inhibitory effect of ellagic Acid on cell growth of ovarian carcinoma cells.

Ellagic acid (EA) is able to inhibit the growth of several cancer cells; however, its effect on human ovarian carcinoma cells has not yet been investigated. Ovarian carcinoma ES-2 and PA-1 cells were treated with EA (10~100 µ M) and assessed for viability, cell cycle, apoptosis, anoikis, autophagy, and chemosensitivity to doxorubicin and their molecular mechanisms. EA inhibited cell proliferation in a dose- and time-dependent manner by arresting both cell lines at the G1 phase of the cell cycle, which were from elevating p53 and Cip1/p21 and decreasing cyclin D1 and E levels. EA also induced caspase-3-mediated apoptosis by increasing the Bax : Bcl-2 ratio and restored anoikis in both cell lines. The enhancement of apoptosis and/or inhibition of autophagy in these cells by EA assisted the chemotherapy efficacy. The results indicated that EA is a potential novel chemoprevention and treatment assistant agent for human ovarian carcinoma.

Induction of apoptosis and cell cycle arrest in human colorectal carcinoma by Litchi seed extract.

The Litchi (Litchi chinensis) fruit products possess rich amounts of flavanoids and proanthocyanidins. Its pericarp has been shown to inhibit breast and liver cancer cell growth. However, the anticolorectal cancer effect of Litchi seed extract has not yet been reported. In this study, the effects of polyphenol-rich Litchi seed ethanol extract (LCSP) on the proliferation, cell cycle, and apoptosis of two colorectal cancer cell lines Colo320DM and SW480 were examined. The results demonstrated that LCSP significantly induced apoptotic cell death in a dose-dependent manner and arrested cell cycle in G2/M in colorectal carcinoma cells. LCSP also suppressed cyclins and elevated the Bax : Bcl-2 ratio and caspase 3 activity. This study provides in vitro evidence that LCSP serves as a potential chemopreventive agent for colorectal cancer.

Effects of ellagic acid on chemosensitivity to 5-fluorouracil in colorectal carcinoma cells.

Ellagic acid has been demonstrated to inhibit the growth of several types of cancer cells. However, whether it sensitizes human colorectal carcinoma cells to 5-fluorouracil, has not yet been investigated. Colorectal carcinoma HT-29, Colo 320DM, SW480 and LoVo cells were treated with ellagic acid (2.5-25 µg/ml) and 5-fluorouracil (5-25 µM) alone and in combination and then the viability was assessed by trypan blue exclusion, apoptosis by annexin-V labeling, mitochondria membrane potential by staining with rhodamine 123, and changes in the levels of proteins involved in apoptosis by immunoblotting. Ellagic acid and 5-fluorouracil synergistically inhibited cell proliferation of HT-29, Colo 320DM and SW480 cells, but cytotoxicity toward LoVo cells seems not to be potentiated by this combination. The combination also elevated apoptotic cell death of HT-29 and Colo 320DM cells. The mitochondria membrane potential was lost in combination-treated HT-29 cells, due to increased B cell lymphoma 2-associated protein X (BAX): B cell lymphoma 2 protein (BCL-2) ratio and caspase-3 activity. Ellagic acid synergistically potentiated chemosensitivity to 5-fluorouracil in at least three colorectal cancer cell lines. The results indicate that ellagic acid has potential as a novel agent sensitizing colorectal cancer cells to 5-fluorouracil.

Inhibitory effect and mechanisms of an anthocyanins- and anthocyanidins-rich extract from purple-shoot tea on colorectal carcinoma cell proliferation.

One newly bred variety of tea cultivar, purple-shoot tea, was selected to evaluate its antiproliferative effects on colorectal carcinoma cells, as well as normal colon cells. The phytochemicals and identified catechins of purple-shoot tea extract (PTE) were significantly higher than that of ordinary tea, especially the anthocyanins (surpassed by 135-fold) and anthocyanidins (surpassed by 3.5-fold). PTE inhibited the proliferation of COLO 320DM (IC(50) = 64.9 µg/mL) and HT-29 (IC(50) = 55.2 µg/mL) by blocking cell cycle progression during the G(0)/G(1) phase and inducing apoptotic death. Western blotting indicated that PTE induced cell cycle arrest by reducing the expression of cyclin E and cyclin D1 in COLO 320DM and the upregulation of p21 and p27 cyclin-dependent kinase inhibitors in HT-29. Two cells treated with PTE also indicated the cleavage of PARP, activation of caspase 3, and an increased Bax/Bcl-2 ratio. Our results showed that PTE is a potential novel dietary agent for colorectal cancer chemoprevention.

Grape-seed procyanidins inhibit the in vitro growth and invasion of pancreatic carcinoma cells.

OBJECTIVES: Grape-seed procyanidins (GSPs) can inhibit cell proliferation and invasiveness in various human cancers. However, the effect of GSP on pancreatic carcinoma cells has not been investigated. METHODS: Pancreatic carcinoma cell lines MIA PaCa-2 and BxPC-3 treated with GSP were assessed for viability by trypan blue exclusion, for cell cycle distribution by flow cytometry, for increased apoptosis by annexin V labeling, for their adhesion and invasion potential by evaluating their ability to penetrate through a matrix gel-coated Boyden chamber, and for changes in the levels of proteins involved in cellular events by immunoblotting. RESULTS: Grape-seed procyanidin inhibited MIA PaCa-2 and BxPC-3 proliferation in a dose-dependent manner and induced G1-phase arrest of the cell cycle in BxPC-3 or mitochondria-mediated apoptosis in MIA PaCa-2. Grape-seed procyanidin also inhibited the adhesion and invasion potential of both cell lines in a dose-dependent manner, which are associated with the suppression of metalloproteases matrix metalloproteinase 9 or 2 (MMP-9 or -2) expression. CONCLUSIONS: Grape-seed procyanidin inhibited the proliferation of pancreatic carcinoma cells by cell cycle blockage or apoptotic induction. The invasiveness was also suppressed by GSP through down-regulation of MMP-2 or MMP-9 in pancreatic carcinoma cells. Grape-seed procyanidin is a potential chemotherapeutic or preventive agent for pancreatic carcinoma.

Potential roles of longan flower and seed extracts for anti-cancer.

Polyphenol-rich plants are known to possess benefits to human health. Recent studies have revealed that many Traditional Chinese Medicines (TCMs) are rich sources of polyphenols and exhibit antioxidant and anti-inflammatory activities, and these TCMs have been shown experimentally to overcome some chronic diseases, including cancer. Longan flowers and seeds, two TCMs traditionally used for relieving pain and urinary diseases, have been revealed in our recent reports and other studies to possess rich amounts of polyphenolic species and exhibit strong anti-oxidant activity, and these could be applied for the treatment of diabetes and cancer. Herein, we review the recent findings regarding the benefits of these two TCMs in the treatment of human cancer and the possible cellular and molecular mechanisms of both substances.

The ACR11 encodes a novel type of chloroplastic ACT domain repeat protein that is coordinately expressed with GLN2 in Arabidopsis.

BACKGROUND: The ACT domain, named after bacterial aspartate kinase, chorismate mutase and TyrA (prephenate dehydrogenase), is a regulatory domain that serves as an amino acid-binding site in feedback-regulated amino acid metabolic enzymes. We have previously identified a novel type of ACT domain-containing protein family, the ACT domain repeat (ACR) protein family, in Arabidopsis. Members of the ACR family, ACR1 to ACR8, contain four copies of the ACT domain that extend throughout the entire polypeptide. Here, we describe the identification of four novel ACT domain-containing proteins, namely ACR9 to ACR12, in Arabidopsis. The ACR9 and ACR10 proteins contain three copies of the ACT domain, whereas the ACR11 and ACR12 proteins have a putative transit peptide followed by two copies of the ACT domain. The functions of these plant ACR proteins are largely unknown. RESULTS: The ACR11 and ACR12 proteins are predicted to target to chloroplasts. We used protoplast transient expression assay to demonstrate that the Arabidopsis ACR11- and ACR12-green fluorescent fusion proteins are localized to the chloroplast. Analysis of an ACR11 promoter-ß-glucuronidase (GUS) fusion in transgenic Arabidopsis revealed that the GUS activity was mainly detected in mature leaves and sepals. Interestingly, coexpression analysis revealed that the GLN2, which encodes a chloroplastic glutamine synthetase, has the highest mutual rank in the coexpressed gene network connected to ACR11. We used RNA gel blot analysis to confirm that the expression pattern of ACR11 is similar to that of GLN2 in various organs from 6-week-old Arabidopsis. Moreover, the expression of ACR11 and GLN2 is highly co-regulated by sucrose and light/dark treatments in 2-week-old Arabidopsis seedlings. CONCLUSIONS: This study reports the identification of four novel ACT domain repeat proteins, ACR9 to ACR12, in Arabidopsis. The ACR11 and ACR12 proteins are localized to the chloroplast, and the expression of ACR11 and GLN2 is highly coordinated. These results suggest that the ACR11 and GLN2 genes may belong to the same functional module. The Arabidopsis ACR11 protein may function as a regulatory protein that is related to glutamine metabolism or signaling in the chloroplast.