Local delivery of carboplatin-loaded hydrogel and calcium carbonate enables two-stage drug release for limited-dose radiation to eliminate mouse malignant glioma.

Postoperative radiotherapy remains the gold standard for malignant glioma treatment. Clinical limitations, including tumor growth between surgery and radiotherapy and the emergence of radioresistance, reduce treatment effectiveness and result in local disease progression. This study aimed to develop a local drug delivery system to inhibit tumor growth before radiotherapy and enhance the subsequent anticancer effects of limited-dose radiotherapy. We developed a compound of carboplatin-loaded hydrogel (CPH) incorporated with carboplatin-loaded calcium carbonate (CPCC) to enable two-stage (peritumoral and intracellular) release of carboplatin to initially inhibit tumor growth and to synergize with limited-dose radiation (10 Gy in a single fraction) to eliminate malignant glioma (ALTS1C1 cells) in a C57BL/6 mouse subcutaneous tumor model. The doses of carboplatin in CPH and CPCC treatments were 150 µL (carboplatin concentration of 5 mg/mL) and 15 mg (carboplatin concentration of 4.1 µg/mg), respectively. Mice receiving the combination of CPH-CPCC treatment and limited-dose radiation exhibited significantly reduced tumor growth volume compared to those receiving double-dose radiation alone. Furthermore, combining CPH-CPCC treatment with limited-dose radiation resulted in significantly longer progression-free survival than combining CPH treatment with limited-dose radiation. Local CPH-CPCC delivery synergized effectively with limited-dose radiation to eliminate mouse glioma, offering a promising solution for overcoming clinical limitations.

Radiosensitizing effects of CDK4/6 inhibitors in hormone receptor-positive and HER2-negative breast cancer mediated downregulation of DNA repair mechanism and NF-κB-signaling pathway.

CDK4/6 inhibitors combined with endocrine therapy prolonged survival in hormone receptor (HR)-positive and HER2-negative advanced breast cancer. We investigated whether CDK4/6 inhibitors enhance radiosensitivity and their underlying mechanisms of this subtype of breast cancer. In vitro and in vivo experiments were conducted using two HR-positive and HER2-negative breast cancer cell lines (MCF-7 and T-47D), CDK4/6 inhibitors (ribociclib and palbociclib) and radiotherapy (RT) to assess the biological functions and mechanisms. The radiation-enhancing effect was assessed using clonogenic assays; γH2AX and 53BP1 levels were assessed by immunofluorescence to evaluate DNA damage. The levels of phospho (p)-ERK, c-Myc, and DNA-double strand break (DSB)-related molecules, p-DNA-PKcs, Rad51, and p-ATM, were assessed by western blotting. We used an NF-κB p65 transcription factor assay kit to evaluate NF-κB activity. We evaluated the antitumor effect of the combination of RT and ribociclib through the MCF-7 orthotopic xenograft model. The synergistic effects of combining RT with ribociclib and palbociclib pretreatment were demonstrated by clonogenic assay. CDK4/6 inhibitors synergistically increased the numbers of RT-induced γH2AX and 53BP1, downregulated the expression of p-DNA-PKcs, Rad51 and p-ATM activated by RT, and reduced RT-triggering p-ERK expression, NF-κB activation, and its down-streaming gene, c-Myc. Combined ribociclib and RT reduced the growth of MCF-7 cell xenograft tumors, and downregulated the immunohistochemical expression of p-ERK, p-NF-κB p65, and c-Myc compared to that in the control group. Combining CDK4/6 inhibitors enhanced radiosensitivity of HR-positive and HER2-negative breast cancer cells at least by reducing DNA-DSB repair and weakening the activation of ERK and NF-κB signaling by RT.

MAP3K1 expression is associated with progression and poor prognosis of hormone receptor-positive, HER2-negative early-stage breast cancer.

PURPOSE: In this study, we assessed whether the overexpression of MAP3K1 promotes the proliferation, migration, and invasion of breast cancer cells, which affect the prognosis of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative early stage breast cancer. METHODS: Two HR-positive, HER2-negative breast cancer cell lines (MCF7 and T-47D) overexpressing MAP3K1 were transfected with two MAP3K1 short hairpin RNA plasmids (shMAP3K1 [#3] and shMAP3K1 [#5]). The proliferation, migration, and invasion of these cells were then examined. We assessed whether shMAP3K1 affects the cell cycle, levels of downstream signaling molecules (ERK, JNK, p38 MAPK, and NF-κB), and sensitivity to chemotherapeutic and hormonal agents. To assess the anti-tumor effect of MAP3K1 knockdown in the breast cancer orthotopic model, MCF7 and T-47D cells treated with or without shMAP3K1 (#3) and shMAP3K1 (#5) were inoculated into the mammary fat pads of mice. In total, 182 patients with HR-positive, HER2-negative T1 and T2 breast cancer and 0-3 nodal metastases were included. Additionally, 73 patients with T1 and T2 breast cancer and negative nodes who received adjuvant endocrine therapy alone were selected as an independent validation cohort. RESULTS: In both cell lines, shMAP3K1 (#3) and shMAP3K1 (#5) significantly reduced cell growth, migration, and invasion by downregulating MMP-9 and by blocking the G2/M phase of the cell cycle and its regulatory molecule cyclin B1. Moreover, both shMAP3K1 (#3) and shMAP3K1 (#5) downregulated ERK-, JNK-, p38 MAPK-, and NF-κB-dependent gene transcription and enhanced the sensitivity of both cell lines to doxorubicin, docetaxel, and tamoxifen. We observed that both shMAP3K1 (#3) and shMAP3K1 (#5) inhibited tumor growth compared with that in the scrambled group of MCF7 and T-47D cell orthotopic tumors. Patients with MAP3K1 overexpression exhibited significantly poorer 10-year disease-free survival (DFS) (70.4% vs. 88.6%, p = 0.003) and overall survival (OS) (81.9% vs. 96.3%, p = 0.001) than those without MAP3K1 overexpression. Furthermore, phospho-ERK (p < 0.001) and phospho-JNK (p < 0.001) expressions were significantly associated with MAP3K1 expression, and both phospho-ERK and phospho-JNK expressions were significantly correlated with poor 10-year DFS and OS. These biological findings, including a significant association between DFS and OS, and the expressions of MAP3K1, phospho-ERK, and phospho-JNK were further validated in an independent cohort. Multivariate analysis identified MAP3K1 expression as an independent poor prognostic factor for DFS and OS. CONCLUSION: Our results indicate that the overexpression of MAP3K1 plays a major role in the poor prognosis of HR-positive, HER2-negative early stage breast cancer.

Atorvastatin Attenuates Radiotherapy-Induced Intestinal Damage through Activation of Autophagy and Antioxidant Effects.

Abdominal or pelvic radiotherapy (RT) often results in small intestinal injury, such as apoptosis of epithelial cells and shortening of the villi. Atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, has many biological effects including cholesterol reduction, protection from cell damage, and autophagy activation. To reduce the extent of radiotherapy- (RT-) induced enteritis, we investigated the protective effects of atorvastatin against RT-induced damage of the intestinal tract. In this study, C57BL/6 mice were randomly distributed into the following groups (n = 8 per group): (1) control group: mice were fed water only, (2) atorvastatin group (Ator): mice were administered atorvastatin, (3) irradiation group (IR): mice received abdominal RT, (4) Ator+IR group: mice received abdominal RT following atorvastatin administration, and (5) Ator+IR+3-MA group: abdominal RT following atorvastatin and 3-methyladenine (an autophagy inhibitor) administration. Based on the assessment of modified Chiu's injury score and villus/crypt ratio, we found that atorvastatin administration significantly reduced intestinal mucosal damage induced by RT. Atorvastatin treatment reduced apoptosis (cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase), DNA damage (γH2AX and 53BP1), oxidative stress (OS, 4-hydroxynonenal), inflammatory molecules (phospho-NF-κB p65 and TGF-ß), fibrosis (collagen I and collagen III), barrier leakage (claudin-2 and fluorescein isothiocyanate-dextran), disintegrity (fatty acid-binding protein 2), and dysfunction (lipopolysaccharide) caused by RT in small intestinal tissue. In addition, atorvastatin upregulated the expression of autophagy-active molecules (LC3B), antioxidants (heme oxygenase 1 and thioredoxin 1), and tight junction proteins (occludin and zonula occludens 1). However, the biological functions of atorvastatin in decreasing RT-induced enteritis were reversed after the administration of 3-MA; the function of antioxidant molecules and activity of thioredoxin reductase were independent of autophagy activation. Our results indicate that atorvastatin can effectively relieve RT-induced enteritis through autophagy activation and associated biological functions, including maintaining integrity and function and decreasing apoptosis, DNA damage, inflammation, OS, and fibrosis. It also acts via its antioxidative capabilities.

Contribution of nuclear BCL10 expression to tumor progression and poor prognosis of advanced and/or metastatic pancreatic ductal adenocarcinoma by activating NF-κB-related signaling.

BACKGROUND: We previously demonstrated that nuclear BCL10 translocation participates in the instigation of NF-κB in breast cancer and lymphoma cell lines. In this study, we assessed whether nuclear BCL10 translocation is clinically significant in advanced and metastatic pancreatic ductal adenocarcinoma (PDAC). METHOD AND MATERIALS: We analyzed the expression of BCL10-, cell cycle-, and NF-κB- related signaling molecules, and the DNA-binding activity of NF-κB in three PDAC cell lines (mutant KRAS lines: PANC-1 and AsPC-1; wild-type KRAS line: BxPC-3) using BCL10 short hairpin RNA (shBCL10). To assess the anti-tumor effect of BCL10 knockdown in PDAC xenograft model, PANC-1 cells treated with or without shBCL10 transfection were inoculated into the flanks of mice. We assessed the expression patterns of BCL10 and NF-κB in tumor cells in 136 patients with recurrent, advanced, and metastatic PDAC using immunohistochemical staining. RESULTS: We revealed that shBCL10 transfection caused cytoplasmic translocation of BCL10 from the nuclei, inhibited cell viability, and enhanced the cytotoxicities of gemcitabine and oxaliplatin in three PDAC cell lines. Inhibition of BCL10 differentially blocked cell cycle progression in PDAC cell lines. Arrest at G1 phase was noted in wild-type KRAS cell lines; and arrest at G2/M phase was noted in mutant KRAS cell lines. Furthermore, shBCL10 transfection downregulated the expression of phospho-CDC2, phospho-CDC25C, Cyclin B1 (PANC-1), Cyclins A, D1, and E, CDK2, and CDK4 (BxPC-3), p-IκBα, nuclear expression of BCL10, BCL3, and NF-κB (p65), and attenuated the NF-κB pathway activation and its downstream molecule, c-Myc, while inhibition of BCL10 upregulated expression of p21, and p27 in both PANC-1 and BxPC-3 cells. In a PANC-1-xenograft mouse model, inhibition of BCL10 expression also attenuated the tumor growth of PDAC. In clinical samples, nuclear BCL10 expression was closely associated with nuclear NF-κB expression (p < 0.001), and patients with nuclear BCL10 expression had the worse median overall survival than those without nuclear BCL10 expression (6.90 months versus 9.53 months, p = 0.019). CONCLUSION: Nuclear BCL10 translocation activates NF-κB signaling and contributes to tumor progression and poor prognosis of advanced/metastatic PDAC.

Maintenance BEZ235 Treatment Prolongs the Therapeutic Effect of the Combination of BEZ235 and Radiotherapy for Colorectal Cancer.

Our previous study demonstrated that administration of NVP-BEZ235 (BEZ235), a dual PI3K/mTOR inhibitor, before radiotherapy (RT) enhanced the radiotherapeutic effect in colorectal cancer (CRC) cells both in vitro and in vivo. Here, we evaluated whether maintenance BEZ235 treatment, after combinatorial BEZ235 + RT therapy, prolonged the antitumor effect in CRC. K-RAS mutant CRC cells (HCT116 and SW480), wild-type CRC cells (HT29), and HCT116 xenograft tumors were separated into the following six study groups: (1) untreated (control); (2) RT alone; (3) BEZ235 alone; (4) RT + BEZ235; (5) maintenance BEZ235 following RT + BEZ235 (RT + BEZ235 + mBEZ235); and (6) maintenance BEZ235 following BEZ235 (BEZ235 + mBEZ235). RT + BEZ235 + mBEZ235 treatment significantly inhibited cell viability and increased apoptosis in three CRC cell lines compared to the other five treatments in vitro. In the HCT116 xenograft tumor model, RT + BEZ235 + mBEZ235 treatment significantly reduced the tumor size when compared to the other five treatments. Furthermore, the expression of mTOR signaling molecules (p-rpS6 and p-eIF4E), DNA double-strand break (DSB) repair-related molecules (p-ATM and p-DNA-PKcs), and angiogenesis-related molecules (VEGF-A and HIF-1α) was significantly downregulated after RT + BEZ235 + mBEZ235 treatment both in vitro and in vivo when compared to the RT + BEZ235, RT, BEZ235, BEZ235 + mBEZ235, and control treatments. Cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), 53BP1, and γ-H2AX expression in the HCT116 xenograft tissue and three CRC cell lines were significantly upregulated after RT + BEZ235 + mBEZ235 treatment. Maintenance BEZ235 treatment in CRC cells prolonged the inhibition of cell viability, enhancement of apoptosis, attenuation of mTOR signaling, impairment of the DNA-DSB repair mechanism, and downregulation of angiogenesis that occurred due to concurrent BEZ235 and RT treatment.

Intratumoral injection of thermogelling and sustained-release carboplatin-loaded hydrogel simplifies the administration and remains the synergistic effect with radiotherapy for mice gliomas.

BACKGROUND: Carboplatin, an antineoplastic agent, binds DNA and enhances radiotherapy (RT) effects. Carboplatin-loaded hydrogel (oxidized hyaluronic acid/adipic acid dihydrazide) enables the sustained drug release and facilitates the synergistic effect with RT. PURPOSE: We investigated the effectiveness and convenience of hydrogel carboplatin combined with RT for mice glioma. MATERIALS AND METHODS: Mouse glioma cells (ALTS1C1) were subcutaneously implanted in the right thigh of C57BL/6 mice on Day 0. The mice were categorized by treatments: sham, hydrogel, hydrogel carboplatin, aqueous carboplatin, RT, hydrogel carboplatin/RT, and aqueous carboplatin/RT. Hydrogel carboplatin (300 µg single dose on Day 7) or aqueous carboplatin (100 µg daily dose on Days 7, 8, and 9) was administered via intratumoral injection. RT was delivered a daily dose of 10 Gy on Days 8 and 9. RESULTS: For mice administered hydrogel carboplatin/RT versus those administered aqueous carboplatin/RT, the 24-day tumor growth control rate and 104-day recurrence-free survival rate were 100% and 50% versus 100% and 66.7% (p = 0.648), respectively. However, mice receiving other treatments showed tumor progression by Day 24 and died within 40 days of tumor cell implantation. CONCLUSIONS: Hydrogel carboplatin simplified intratumoral drug delivery and remained the synergistic effects with RT, which is potential for clinical applications.

First-line antibiotic therapy in Helicobacter pylori-negative low-grade gastric mucosa-associated lymphoid tissue lymphoma.

First-line antibiotic treatment for eradicating Helicobacter pylori (HP) infection is effective in HP-positive low-grade gastric mucosa-associated lymphoid tissue lymphoma (MALToma), but its role in HP-negative cases is uncertain. In this exploratory retrospective study, we assessed the outcome and potential predictive biomarkers for 25 patients with HP-negative localized gastric MALToma who received first-line HP eradication (HPE) therapy. An HP-negative status was defined as negative results on histology, rapid urease test, 13C urea breath test, and serology. We observed an antibiotic response (complete remission [CR], number = 8; partial remission, number = 1) in 9 (36.0%) out of 25 patients. A t(11;18)(q21;q21) translocation was detected in 7 (43.8%) of 16 antibiotic-unresponsive cases, but in none of the 9 antibiotic-responsive cases (P = 0.027). Nuclear BCL10 expression was significantly higher in antibiotic-unresponsive tumors than in antibiotic-responsive tumors (14/16 [87.5%] vs. 1/9 [11.1%]; P = 0.001). Nuclear NF-κB expression was also significantly higher in antibiotic-unresponsive tumors than in antibiotic-responsive tumors (12/16 [75.0%] vs. 1/9 [11.1%]; P = 0.004). A substantial portion of patients with HP-negative gastric MALToma responded to first-line HPE. In addition to t(11;18)(q21;q21), BCL10 and NF-κB are useful immunohistochemical biomarkers to predict antibiotic-unresponsive status in this group of tumors.

The B-cell-activating factor signalling pathway is associated with Helicobacter pylori independence in gastric mucosa-associated lymphoid tissue lymphoma without t(11;18)(q21;q21).

We previously reported that activation of the B-cell-activating factor (BAFF) pathway upregulates nuclear factor-κB (NF-κB) and induces BCL3 and BCL10 nuclear translocation in Helicobacter pylori (HP)-independent gastric diffuse large B-cell lymphoma (DLBCL) tumours with evidence of mucosa-associated lymphoid tissue (MALT). However, the significance of BAFF expression in HP independence of gastric low-grade MALT lymphomas without t(11;18)(q21;q21) remains unexplored. Sixty-four patients who underwent successful HP eradication for localized HP-positive gastric MALT lymphomas without t(11;18)(q21;q21) were studied. BAFF expression was significantly higher in the HP-independent group than in the HP-dependent group [22/26 (84.6%) versus 8/38 (21.1%); p < 0.001]. Similarly, BAFF receptor (BAFF-R) expression (p = 0.004) and nuclear BCL3 (p = 0.004), BCL10 (p < 0.001), NF-κB (p65) (p = 0.001) and NF-κB (p52) (p = 0.005) expression were closely correlated with the HP independence of these tumours. Moreover, BAFF overexpression was significantly associated with BAFF-R expression and nuclear BCL3, BCL10, NF-κB (p65) and NF-κB (p52) expression. These findings were further validated in an independent cohort, including 40 HP-dependent cases and 18 HP-independent cases of gastric MALT lymphoma without t(11;18)(q21;q21). The biological significance of BAFF signalling in t(11;18)(q21;q21)-negative lymphoma cells was further studied in two types of lymphoma B cell: OCI-Ly3 [non-germinal centre B-cell origin DLBCL without t(11;18)(q21;q21) cell line] and MA-1 [t(14;18)(q32;q21)/IGH-MALT1-positive DLBCL cell line]. In both cell lines, we found that BAFF activated the canonical NF-κB and AKT pathways, and induced the formation of BCL10-BCL3 complexes, which translocated to the nucleus. BCL10 and BCL3 nuclear translocation and NF-κB (p65) transactivation were inhibited by either LY294002 or by silencing BCL3 or BCL10 with small interfering RNA. BAFF also activated non-canonical NF-κB pathways (p52) through tumour necrosis factor receptor-associated factor 3 degradation, NF-κB-inducing kinase accumulation, inhibitor of κB kinase (IKK) α/ß phosphorylation and NF-κB p100 processing in both cell lines. Our data indicate that the autocrine BAFF signal transduction pathway contributes to HP independence in gastric MALT lymphomas without the t(11;18)(q21;q21) translocation. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Targeting Colorectal Cancer Stem-Like Cells with Anti-CD133 Antibody-Conjugated SN-38 Nanoparticles.

Cancer stem-like cells play a key role in tumor development, and these cells are relevant to the failure of conventional chemotherapy. To achieve favorable therapy for colorectal cancer, PEG-PCL-based nanoparticles, which possess good biological compatibility, were fabricated as nanocarriers for the topoisomerase inhibitor, SN-38. For cancer stem cell therapy, CD133 (prominin-1) is a theoretical cancer stem-like cell (CSLC) marker for colorectal cancer and is a proposed therapeutic target. Cells with CD133 overexpression have demonstrated enhanced tumor-initiating ability and tumor relapse probability. To resolve the problem of chemotherapy failure, SN-38-loaded nanoparticles were conjugated with anti-CD133 antibody to target CD133-positive (CD133(+)) cells. In this study, anti-CD133 antibody-conjugated SN-38-loaded nanoparticles (CD133Ab-NPs-SN-38) efficiently bound to HCT116 cells, which overexpress CD133 glycoprotein. The cytotoxic effect of CD133Ab-NPs-SN-38 was greater than that of nontargeted nanoparticles (NPs-SN-38) in HCT116 cells. Furthermore, CD133Ab-NPs-SN-38 could target CD133(+) cells and inhibit colony formation compared with NPs-SN-38. In vivo studies in an HCT116 xenograft model revealed that CD133Ab-NPs-SN-38 suppressed tumor growth and retarded recurrence. A reduction in CD133 expression in HCT116 cells treated with CD133Ab-NPs-SN-38 was also observed in immunohistochemistry results. Therefore, this CD133-targeting nanoparticle delivery system could eliminate CD133-positive cells and is a potential cancer stem cell targeted therapy.

A theranostic micelleplex co-delivering SN-38 and VEGF siRNA for colorectal cancer therapy.

The development of an efficient colorectal cancer therapy is currently a public health priority. In the present work, we proposed a multifunctional theranostic micellar drug delivery system utilizing cationic PDMA-block-poly(ε-caprolactone) (PDMA-b-PCL) micelles as nanocarriers of SN-38 (7-ethyl-10-hydroxycamptothecin), ultra-small superparamagnetic iron oxide nanoparticles (USPIO), and small interfering RNA (siRNA) that targets human vascular endothelial growth factor (VEGF). The VEGF siRNA was conjugated to polyethylene glycol (PEG) (siRNA-PEG) before complexation with the micelles in order to improve the siRNA's stability and to prolong its retention time in the blood circulation. To further improve the in vivo biosafety, we prepared mixed micelles using mPEG-PCL together with PDMA-b-PCL copolymer. The SN-38/USPIO-loaded siRNA-PEG mixed micelleplexes passively targeted to tumor regions and synergistically facilitated VEGF silencing and chemotherapy, thus efficiently suppressing tumor growth via a multi-dose therapy regimen. Additionally, the SN-38/USPIO-loaded siRNA-PEG mixed micelleplexes acted as a negative magnetic resonance imaging (MRI) contrast agent in T2-weighted imaging, resulting in a powerful tool for the diagnosis and for tracking of the therapeutic outcomes. In summary, we established a theranostic micellar drug and gene delivery system that not only synergistically combined gene silencing and chemotherapy but also served as a negative MRI contrast agent, which reveal its potential as a novel colorectal cancer therapy.

Dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor is an effective radiosensitizer for colorectal cancer.

The present study was aimed to investigate whether combination of molecular targeting therapy, a dual PI3K/mTOR inhibitor (BEZ235), with radiation can enhance the radiosensitivity of colorectal cancer cells (CRC). K-RAS mutant CRC cells (HCT 116 and SW 620) and wild type CRC cells (HT 29) were irradiated with different dose of radiation (0-6 Gy). The synergistic effects of combining radiation with different concentration of BEZ235 (0-10 nM) pretreatment were demonstrated by cell survival assay. When comparing with radiation alone and BEZ235 alone, the combination of BEZ235 pretreatment and radiation resulted in an increased percentage of sub-G1 phase cells, and an increased number of γ-H2AX/cell (DNA double strand breaks). Radiation up-regulated AKT/mTOR signaling pathway, including the activation of phospho (p)-AKT, p-mTOR, p-eIF4E, and p-rpS6; and this activated AKT/mTOR signaling pathway was attenuated by BEZ235 pretreatment. In addition, BEZ235 blocked double strand break repair induced by radiation through attenuating the activation of ATM and DNA-PKcs and sensitized CRC cells to radiation. In vivo model, the tumor size and the expression pattern of p-mTOR, p-eIF4E, and p-rpS6 were significantly decreased in combined group than radiation alone or BEZ235 alone. Our findings indicate that the administration of BEZ235 before radiation enhances the radiotherapeutic effect of CRC cells both in vitro and in vivo.

Depletion of arginine by recombinant arginine deiminase induces nNOS-activated neurotoxicity in neuroblastoma cells.

The abnormal regulation of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS) is associated with neurodegenerative disorders. Recombinant arginine deiminase (rADI) is a selective NO modulator of iNOS and eNOS in endothelial cells, and it also exhibits neuroprotective activity in an iNOS-induced neuron-microglia coculture system. However, the effect of rADI on nNOS remains unknown. Addressing this issue is important for evaluating the potential application of rADI in neurodegenerative diseases. SH-SY5Y cells were treated with N-methyl-D-aspartic acid (NMDA) to activate nNOS. NMDA increased NO production by 39.7 ± 3.9% via nNOS under arginine-containing conditions, but there was no significant increase in both arginine-free and rADI pretreated arginine-containing (citrulline) buffer. Subsequently, neither NMDA nor rADI alone caused cytotoxicity, whereas cotreatment with NMDA and rADI resulted in dissipation of the cell mitochondrial membrane potential and decreased cell viability. The mechanism of rADI cytotoxicity in the presence of NMDA is caused by the inhibition of NO production via nNOS mediated by the NMDA receptor, which was abolished when extracellular arginine was absent, even in the presence of citrulline. rADI not only reduced NO production but also caused cellular toxicity in nNOS-activated SH-SY5Y cells, suggesting a dual role for rADI in NOS-mediated neurotoxicity.

Autophagy promotes resistance to photodynamic therapy-induced apoptosis selectively in colorectal cancer stem-like cells.

Recent studies have indicated that cancer stem-like cells (CSCs) exhibit a high resistance to current therapeutic strategies, including photodynamic therapy (PDT), leading to the recurrence and progression of colorectal cancer (CRC). In cancer, autophagy acts as both a tumor suppressor and a tumor promoter. However, the role of autophagy in the resistance of CSCs to PDT has not been reported. In this study, CSCs were isolated from colorectal cancer cells using PROM1/CD133 (prominin 1) expression, which is a surface marker commonly found on stem cells of various tissues. We demonstrated that PpIX-mediated PDT induced the formation of autophagosomes in PROM1/CD133(+) cells, accompanied by the upregulation of autophagy-related proteins ATG3, ATG5, ATG7, and ATG12. The inhibition of PDT-induced autophagy by pharmacological inhibitors and silencing of the ATG5 gene substantially triggered apoptosis of PROM1/CD133(+) cells and decreased the ability of colonosphere formation in vitro and tumorigenicity in vivo. In conclusion, our results revealed a protective role played by autophagy against PDT in CSCs and indicated that targeting autophagy could be used to elevate the PDT sensitivity of CSCs. These findings would aid in the development of novel therapeutic approaches for CSC treatment.

Intracellular delivery of recombinant arginine deiminase (rADI) by heparin-binding hemagglutinin adhesion peptide restores sensitivity in rADI-resistant cancer cells.

Recombinant arginine deiminase (rADI) has been used in clinical trials for arginine-auxotrophic cancers. However, the emergence of rADI resistance, due to the overexpression of argininosuccinate synthetase (AS), has introduced an obstacle in its clinical application. Here, we have proposed a strategy for the intracellular delivery of rADI, which depletes both extracellular and intracellular arginine, to restore the sensitivity of rADI-resistant cancer cells. In this study, the C terminus of heparin-binding hemagglutinin adhesion protein from Mycobacterium tuberculosis (HBHAc), which contains 23 amino acids, was used to deliver rADI into rADI-resistant human breast adenocarcinoma cells (MCF-7). Chemical conjugates (l- and d-HBHAc-SPDP-rADI) and a recombinant fusion protein (rHBHAc-ADI) were produced. l- and d-HBHAc-SPDP-rADI showed a significantly higher cellular uptake of rADI by MCF-7 cells compared to that of rADI alone. Cell viability was significantly decreased in a dose-dependent manner in response to l- and d-HBHAc-SPDP-rADI treatments. In addition, the ratio of intracellular concentration of citrulline to arginine in cells treated with l- and d-HBHAc-SPDP-rADI was significantly increased by 1.4- and 1.7-fold, respectively, compared with that obtained in cells treated with rADI alone (p < 0.001). Similar results were obtained with the recombinant fusion protein rHBHAc-ADI. Our study demonstrates that the increased cellular uptake of rADI by HBHAc modification can restore the sensitivity of rADI treatment in MCF-7 cells. rHBHAc-ADI may represent a novel class of antitumor enzyme with an intracellular mechanism that is independent of AS expression.

Simultaneous detection of single nucleotide polymorphisms and copy number variations in the CYP2D6 gene by multiplex polymerase chain reaction combined with capillary electrophoresis.

CYP2D6 (cytochrome P450 2D6) is one of the most important enzymes involved in drug metabolism, and CYP2D6 gene variants may cause toxic effects of therapeutic drugs or treatment failure. In this research, a rapid and simple method for genotyping the most common mutant alleles in the Asian population (CYP2D6*1/*1, CYP2D6*1/*10, CYP2D6*10/*10, CYP2D6*1/*5, CYP2D6*5/*10, and CYP2D6*5/*5) was developed by allele-specific polymerase chain reaction (AS-PCR) combined with capillary electrophoresis (CE). We designed a second mismatch nucleotide next to the single nucleotide polymorphism (SNP) site in allele-specific primers to increase the difference in PCR amplification. Besides, we established simulation equations to predict the CYP2D6 genotypes by analyzing the DNA patterns in the CE chromatograms. The multiplex PCR combined with CE method was applied to test 50 patients, and all of the test results were compared with the DNA sequencing method, long-PCR method and real-time PCR method. The correlation of the analytical results between the proposed method and other methods were higher than 90%, and the proposed method is superior to other methods for being able to simultaneous detection of SNPs and copy number variations (CNV). Furthermore, we compared the plasma concentration of aripiprazole (a CYP2D6 substrate) and its major metabolites with the genotype of 25 patients. The results demonstrate the proposed genotyping method is effective for estimating the activity of the CYP2D6 enzyme and shows potential for application in personalized medicine. Similar approach can be applied to simultaneous detection of SNPs and CNVs of other genes.

Combined effects of peroxisome proliferator-activated receptor alpha and apolipoprotein E polymorphisms on risk of breast cancer in a Taiwanese population.

BACKGROUND: The peroxisome proliferator-activated receptor alpha (PPARA) and apolipoprotein E (APOE) proteins are reported to be correlated with lipid metabolism, cardiovascular disease, and breast cancer. METHODS: We screened APOE and PPARA (S24F and V227A) polymorphisms in 306 breast cancer patients and 300 noncancer controls and determined the relationship between their genetic polymorphisms and breast cancer risk. Interactions with clinical characteristics were also examined. RESULTS: We found that the risk of breast cancer was associated with APOE genotypes (P = 0.014) but not with PPARA S24F or V227A genotypes. The combined effects of F24/APOE genotypes (P = 0.003) on breast cancer risk were more significant than the individual effect of APOE genotypes (P = 0.014). F24/ε4 carriers had a higher tendency to develop breast cancer than F24/ε3 carriers (P = 0.013), and this effect is stronger than with individual ε4 carriers (P = 0.029). In addition, both F24/ε4 and V227/ε4 carriers were significantly enriched in the human epidermal growth factor receptor 2/neu negative status. CONCLUSIONS: These findings suggest that the APOE ε4 genotype plays a major role in the prediction of breast cancer, but the PPARA F24 mutation enhances this outcome. The combined effects of F24/ε4 genotypes are positively associated with risk of breast cancer.

RNA interference of argininosuccinate synthetase restores sensitivity to recombinant arginine deiminase (rADI) in resistant cancer cells.

BACKGROUND: Sensitivity of cancer cells to recombinant arginine deiminase (rADI) depends on expression of argininosuccinate synthetase (AS), a rate-limiting enzyme in synthesis of arginine from citrulline. To understand the efficiency of RNA interfering of AS in sensitizing the resistant cancer cells to rADI, the down regulation of AS transiently and permanently were performed in vitro, respectively. METHODS: We studied the use of down-regulation of this enzyme by RNA interference in three human cancer cell lines (A375, HeLa, and MCF-7) as a way to restore sensitivity to rADI in resistant cells. The expression of AS at levels of mRNA and protein was determined to understand the effect of RNA interference. Cell viability, cell cycle, and possible mechanism of the restore sensitivity of AS RNA interference in rADI treated cancer cells were evaluated. RESULTS: AS DNA was present in all cancer cell lines studied, however, the expression of this enzyme at the mRNA and protein level was different. In two rADI-resistant cell lines, one with endogenous AS expression (MCF-7 cells) and one with induced AS expression (HeLa cells), AS small interference RNA (siRNA) inhibited 37-46% of the expression of AS in MCF-7 cells. ASsiRNA did not affect cell viability in MCF-7 which may be due to the certain amount of residual AS protein. In contrast, ASsiRNA down-regulated almost all AS expression in HeLa cells and caused cell death after rADI treatment. Permanently down-regulated AS expression by short hairpin RNA (shRNA) made MCF-7 cells become sensitive to rADI via the inhibition of 4E-BP1-regulated mTOR signaling pathway. CONCLUSIONS: Our results demonstrate that rADI-resistance can be altered via AS RNA interference. Although transient enzyme down-regulation (siRNA) did not affect cell viability in MCF-7 cells, permanent down-regulation (shRNA) overcame the problem of rADI-resistance due to the more efficiency in AS silencing.