Discovery of novel EGFR-PROTACs capable of degradation of multiple EGFR-mutated proteins.

Although three generations of Epidermal growth factor receptor (EGFR) - TK inhibitors have been approved for the treatment of Non-small-cell lung cancers (NSCLC), their clinical application is still largely hindered by acquired drug resistance mediated new EGFR mutations and side effects. The Proteolysis targeting chimera (PROTAC) technology has the potential to overcome acquired resistance from mutant EGFR through a novel mechanism of action. In this study, we developed the candidate degrader IV-3 by structural modifications of the lead compound 13, which exhibited limited antiproliferative activity against HCC-827 cells. Compared to compound 13, IV-3 exhibited remarkable anti-proliferative activity against HCC-827 cells, NCI-H1975 cells, and NCI-H1975-TM cells (IC50 = 0.009 µM, 0.49 µM and 3.24 µM, respectively), as well as significantly inducing degradation of EGFR protein in these cell lines (DC50 = 17.93 nM, 0.25 µM and 0.63 µM, respectively). Further investigations confirmed that IV-3 exhibited superior anti-tumor activity in all xenograft tumor models through the degradation of mutant EGFR protein. Moreover, IV-3 showed no inhibitory activity against A431 and A549 cells expressing wild-type EGFR, thereby eliminating potential toxic side effects emerging from wild-type EGFR inhibition. Overall, our study provides promising insights into EGFR-PROTACs as a potential therapeutic strategy against EGFR-acquired mutation.

Vascularization ability of glioma stem cells in different three-dimensional microenvironments.

Glioblastoma (GBM) is among the most common and aggressive adult central nervous system tumors. One prominent characteristic of GBM is the presence of abnormal microvessels. A significant correlation between angiogenesis and prognosis has been observed. Accurately reconstructing this neovascularization and tumor microenvironment through personalized in vitro disease models presents a significant challenge. However, it is crucial to develop new anti-angiogenic therapies for GBM. In this study, 3D bioprinted glioma stem cell (GSC)-laden hydrogel scaffolds, hybrid GSC hydrogels and cell-free hydrogel scaffolds were manufactured to investigate the vascularization ability of GSCs in varying 3D microenvironments. Our results demonstrated that the bioactivity of GSCs in the 3D bioprinted GSC-laden hydrogel scaffold was preferable and stable, and the amounts of vascular endothelial growth factor A and basic fibroblast growth factor were the highest in the microenvironment. When the three different models were co-cultured with human umbilical vein endothelial cells, the expression of angiogenesis-related markers was the most abundant in the bioprinted GSC-laden hydrogel scaffold. Additionally, xenograft tumors formed by bioprinted GSC-laden hydrogel scaffolds more closely resembled human gliomas regarding color, texture and vascularization. Notably, in xenograft tumors derived from 3D bioprinted GSC-laden hydrogel scaffolds, the number of human CD105+ cells was significantly higher, and human endothelial vascular lumen-like structures were observed. This indicates that the 3D bioprinted GSC-laden hydrogel scaffold is a suitable model for mimicking the glioma microenvironment and studying tumor angiogenesis.

Luciferase Expressing Preclinical Model Systems Representing the Different Molecular Subtypes of Colorectal Cancer.

Colorectal cancer (CRC) is a heterogeneous disease. More insight into the biological diversity of CRC is needed to improve therapeutic outcomes. Established CRC cell lines are frequently used and were shown to be representative models of the main subtypes of CRC at the genomic and transcriptomic level. In the present work, we established stable, luciferase expressing derivatives from 10 well-established CRC cell lines, generated spheroids and subcutaneous xenograft tumors in nude mice, and performed comparative characterization of these model systems. Transcriptomic analyses revealed the close relation of cell lines with their derived spheroids and xenograft tumors. The preclinical model systems clustered with patient tumor samples when compared to normal tissue thereby confirming that cell-line-based tumor models retain specific characteristics of primary tumors. Xenografts showed different differentiation patterns and bioluminescence imaging revealed metastatic spread to the lungs. In addition, the models were classified according to the CMS classification system, with further sub-classification according to the recently identified two intrinsic epithelial tumor cell states of CRC, iCMS2 and iCMS3. The combined data showed that regarding primary tumor characteristics, 3D-spheroid cultures resemble xenografts more closely than 2D-cultured cells do. Furthermore, we set up a bioluminescence-based spheroid cytotoxicity assay in order to be able to perform dose-response relationship studies in analogy to typical monolayer assays. Applying the established assay, we studied the efficacy of oxaliplatin. Seven of the ten used cell lines showed a significant reduction in the response to oxaliplatin in the 3D-spheroid model compared to the 2D-monolayer model. Therapy studies in selected xenograft models confirmed the response or lack of response to oxaliplatin treatment. Analyses of differentially expressed genes in these models identified CAV1 as a possible marker of oxaliplatin resistance. In conclusion, we established a combined 2D/3D, in vitro/in vivo model system representing the heterogeneity of CRC, which can be used in preclinical research applications.

The Interaction Between SMAD1 and YAP1 Is Correlated with Increased Resistance of Gastric Cancer Cells to Cisplatin.

Drug resistance is a major obstacle leading to treating failure and poor outcome in gastric cancer (GC). This study explores the interaction between SMAD family member 1 (SMAD1) and Yes1-associated transcriptional regulator (YAP1) and their roles in cisplatin (DDP) resistance in GC. Transcriptome analysis predicted that SMAD1 is highly expressed in DDP-resistant cells. Elevated SMAD1 expression was detected in GC tissue and cells, especially in DDP-resistant cells (MKN-45/DDP and AGS/DDP). SMAD1 downregulation in cells decreased 50% inhibition value of DDP, reduced proliferation, migration, and invasion, and promoted cell cycle arrest and apoptosis. A protein-protein interaction network suggested a possible SMAD1 and YAP1 interaction in GC. The SMAD1 and YAP1 interaction was validated by chromatin immunoprecipitation (ChIP), co-immunoprecipitation (Co-IP), and luciferase assays. SMAD1 bound to YAP1 and activated its transcription. SMAD1 formed complexes with YAP1 in nucleus, and YAP1 upregulation enhanced SMAD1 activity as well. Upregulation of YAP1 restored the malignant behaviors of GC cells suppressed by SMAD1 silencing. In vivo, SMAD1 silencing suppressed growth and DDP resistance of xenograft tumors in nude mice, and this suppression was blocked by YAP1 overexpression again. In conclusion, this study demonstrates that SMAD1 can interact with YAP1 to enhance the DDP resistance of GC cells.

Glucose Conferred Irinotecan Chemoresistance through Divergent Actions of Pyruvate and ATP in Cell Death and Proliferation of Colorectal Cancer.

BACKGROUND: Altered glucose metabolism is associated with chemoresistance in colorectal cancer (CRC). This study aimed to illustrate the molecular mechanisms of glucose-mediated chemoresistance against irinotecan, a topoisomerase I inhibitor, focusing on the distinct roles of metabolites such as pyruvate and ATP in modulating cell death and proliferation. METHODS: Four human CRC cell lines, tumorspheres, and mouse xenograft models were treated with various doses of irinotecan in the presence of various concentrations of glucose, pyruvate, or ATP-encapsulated liposomes. RESULTS: In this study, human CRC cell lines treated with irinotecan in high glucose displayed increased cell viability and larger xenograft tumor sizes in mouse models compared to those treated in normal glucose concentrations. Irinotecan induced apoptosis and necroptosis, both mitigated by high glucose. Liposomal ATP prevented irinotecan-induced apoptosis, while it did not affect necroptosis. In contrast, pyruvate attenuated the receptor-interacting protein kinase 1/3-dependent necroptosis via free radical scavenging without modulating apoptotic levels. Regarding the cell cycle, liposomal ATP aggravated the irinotecan-induced G0/G1 shift, whereas pyruvate diminished the G0/G1 shift, showing opposite effects on proliferation. Last, tumorsphere structural damage, an index of solid tumor responsiveness to chemotherapy, was determined. Liposomal ATP increased tumorsphere size while pyruvate prevented the deformation of spheroid mass. CONCLUSIONS: Glucose metabolites confer tumor chemoresistance via multiple modes of action. Glycolytic pyruvate attenuated irinotecan-induced necroptosis and potentiated drug insensitivity by shifting cells from a proliferative to a quiescent state. On the other hand, ATP decreased irinotecan-induced apoptosis and promoted active cell proliferation, contributing to tumor recurrence. Our findings challenged the traditional view of ATP as the main factor for irinotecan chemoresistance and provided novel insights of pyruvate acting as an antioxidant responsible for drug insensitivity, which may shed light on the development of new therapies against recalcitrant cancers.

Curcumol inhibits the growth of xenograft-tumors in mice and the biological activities of pancreatic cancer cells by regulating the miR-21-5p/SMAD7 axis.

Anti-cancer effects of curcumol on various cancers have been reported previously. This study focused on investigating the role of curcumol in pancreatic cancer from the molecular perspective. The survival of pancreatic cancer patients with high or low expression of miR-21-5pand the target gene of miR-21-5pwere analyzed by bioinformatics. MiR-21-5p expression in cancer tissues was analyzed by RT-qPCR. Anxenograft-tumor BALB/c nude mice model was established and pancreatic cancer cells were cultured. Later, the mice and cells were further treated with curcumol. The tumor size and weightas well as mice body weight were recorded. The viability, proliferation, migration, and invasion of the cells were evaluated by MTT, colony formation, and transwell assays, respectively. The expressions of molecules in the xenograft-tumor tissues or cells were detected by immunohistochemical assay, Western blot, or RT-qPCR. MiR-21-5p was high-expressed in pancreatic cancer tissues and patients with high expression of miR-21-5p had poor survival. Curcumol inhibited the xenograft-tumor size, tumor weight, and PCNA and miR-21-5p expressions while promoting Cleaved caspase-3 expression in xenograft-tumor tissues. Curcumol inhibited the viability, proliferation, migration, invasion, and miR-21-5p expression, but increased SMAD7 expression in cancer cells. MiR-21-5p overexpression reversed the effect of curcumol on cancer cells, and decreased the E-cadherin expression while elevating the expressions of PCNA, N-cadherin, Vimentin, p-SMAD2, and p-SMAD3 in curcumol-treated cells. The overexpression of SMAD7, a target gene of miR-21-5p, reversed the effect of miR-21-5p on curcumol-treated cells. Curcumol inhibited growth of xenograft-tumors and the biological activities of pancreatic cancer cells by regulating the miR-21-5p/SMAD7 axis.

Iso-suillin-induced DNA damage leading to cell cycle arrest and apoptosis arised from p53 phosphorylation in A549 cells.

Extensive investigations have revealed that iso-suillin, a secondary metabolite isolated from Suillus flavus, could induce cell cycle arrest and apoptosis in human chronic myeloid leukemia K562 cells, human hepatocellular carcinoma SMMC-7721 cell line, and human small cell lung cancer H446 cell line in vitro. In the present study, human lung cancer A549 cells were used to reveal the mechanism of iso-suillin's effects on lung adenocarcinoma, which were detected both in vitro and in vivo. Results showed that iso-suillin potently inhibited A549 cell proliferation through an early G1 arrest. Iso-suillin also induced A549 cell apoptosis in vitro. Phosphorylation of p53 at serines 15 and 20 may be one of the pivotal factors for cell cycle arrest and apoptosis after treatment of iso-suillin in A549 cells. Moreover, in an A549 xenograft model, tumor growth and progression could be inhibited by iso-suillin. Body weight change and some vital organs toxicity was also roughly examined, no significant toxic effects of iso-suillin were shown (at a dose of 5 mg/kg for each administration). The in vitro and in vivo anti-tumor effects implied that iso-suillin may act as a tumor growth inhibitor, and its induction of p53 phosphorylation is pivotal for cell cycle arrest and apoptosis in A549 cells.

TET2 and DNMT3A mutations and exceptional response to 4'-thio-2'-deoxycytidine in human solid tumor models.

BACKGROUND: Challenges remain on the selection of patients who potentially respond to a class of drugs that target epigenetics for cancer treatment. This study aims to investigate TET2/DNMT3A mutations and antitumor activity of a novel epigenetic agent in multiple human cancer cell lines and animal models. METHODS: Seventeen cancer cell lines and multiple xenograft models bearing representative human solid tumors were subjected to 4'-thio-2'-deoxycytidine (T-dCyd) or control treatment. Gene mutations in cell lines were examined by whole exome and/or Sanger sequencing. Specific gene expression was measured in cells and xenograft tumor samples by Western blotting and immunohistochemistry. TET2/DNMT3A mutation status in 47,571 human tumor samples was analyzed at cBioPortal for Cancer Genomics. RESULTS: Cell survival was significantly inhibited by T-dCyd in breast BT549, lung NCI-H23, melanoma SKMEL5 and renal ACHN cancer lines harboring deleterious TET2 and nonsynonymous DNMT3A mutations compared to 13 lines without such mutation pattern (P = 0.007). The treatment upregulated p21 and induced cell cycle arrest in NCI-H23 cells, and dramatically inhibited their xenograft tumor growth versus wildtype models. T-dCyd administrations led to a significant p21 increase and near eradication of tumor cells in the double-mutant xenografts by histological evaluation. TET2/DNMT3A was co-mutated in human lung, breast, skin and kidney cancers and frequently in angioimmunoblastic and peripheral T cell lymphomas and several types of leukemia. CONCLUSIONS: Cell and animal models with concurrent mutations in TET2 and DNMT3A were sensitive to T-dCyd treatment. The mutations were detectable in human solid tumors and frequently occur in some hematological malignancies.

Nelfinavir Inhibits the Growth of Small-cell Lung Cancer Cells and Patient-derived Xenograft Tumors.

BACKGROUND/AIM: Small-cell lung cancer (SCLC) is aggressive and confers poor prognosis. Although SCLC shows more response to chemotherapy than other types of lung cancer, it is difficult to cure because of its frequent recurrence. New drugs and molecular targets need to be identified. MATERIALS AND METHODS: We investigated the effect of nelfinavir, an HIV protease inhibitor, on SCLC cells and in preclinical treatment studies using SCLC patient-derived xenograft (PDX) mouse models. RESULTS: Nelfinavir inhibited SCLC cell proliferation and induced cell death in vitro, which was caused by induction of the unfolded protein response (UPR), inhibition of mammalian/mechanistic target of rapamycin (mTOR) activation, and reduction in the expression of SCLC-related molecules such as achaete-scute homolog 1 (ASCL1). In vivo, nelfinavir inhibited the growth of SCLC PDX tumors, which correlated with the induction of UPR and reduced expression of ASCL1. CONCLUSION: Nelfinavir is highly effective in SCLC in vitro and in vivo, suggesting possible incorporation of nelfinavir into clinical trials for patients with SCLC.

Identification of Tumor Antigens in the HLA Peptidome of Patient-derived Xenograft Tumors in Mouse.

Personalized cancer immunotherapy targeting patient-specific cancer/testis antigens (CTA) and neoantigens may benefit from large-scale tumor human leukocyte antigen (HLA) peptidome (immunopeptidome) analysis, which aims to accurately identify antigens presented by tumor cells. Although significant efforts have been invested in analyzing the HLA peptidomes of fresh tumors, it is often impossible to obtain sufficient volumes of tumor tissues for comprehensive HLA peptidome characterization. This work attempted to overcome some of these obstacles by using patient-derived xenograft tumors (PDX) in mice as the tissue sources for HLA peptidome analysis. PDX tumors provide a proxy for the expansion of the patient tumor by re-grafting them through several passages to immune-compromised mice. The HLA peptidomes of human biopsies were compared with those derived from PDX tumors. Larger HLA peptidomes were obtained from the significantly larger PDX tumors as compared with the patient biopsies. The HLA peptidomes of different PDX tumors derived from the same source tumor biopsy were very reproducible, even following subsequent passages to new naïve mice. Many CTA-derived HLA peptides were discovered, as well as several potential neoantigens/variant sequences. Taken together, the use of PDX tumors for HLA peptidome analysis serves as a highly expandable and stable source of reproducible and authentic peptidomes, opening up new opportunities for defining large HLA peptidomes when only small tumor biopsies are available. This approach provides a large source for tumor antigens identification, potentially useful for personalized immunotherapy.

Immunohistochemical Analysis of Transcription Factors and Markers of Epithelial-Mesenchymal Transition (EMT) in Human Tumors.

BACKGROUND/AIM: Epithelial-mesenchymal transition (EMT) is a key multi-step process which enables cancer cells to detach from the epithelial primary tumor mass and allows them to metastasize to distant organs. We immunohistochemically analyzed the expression of the transcription factors (TWIST-1, SLUG, ZEB1, ZEB2) and components of the extracellular matrix (laminin-5, fibronectin) which influence the EMT. MATERIALS AND METHODS: Primary human breast (MDA-MB-231), colon (HT29, HCT116), ovarian (SKOV3, OVCAR3) and head and neck squamous cell carcinoma cell lines (UTSCC2, UTSCC24A) grown as xenografts were immunohistochemically analyzed in vitro and in vivo. RESULTS: A high SLUG expression was observed in every cancer entity both in vitro and in vivo. ZEB1 and ZEB2 showed a high in vivo expression especially in SKOV3 and in in vitro grown MDA-MB-231 cells. CONCLUSION: SLUG expression showed the highest expression in all cancer entities investigated. Hence, it presumably represents the master regulator of EMT in these metastatic tumor entities.

Mesenchymal stem cells confer chemoresistance in breast cancer via a CD9 dependent mechanism.

The development of chemotherapy drug resistance remains a significant barrier for effective therapy in several cancers including breast cancer. Bone marrow-derived mesenchymal stem cells (BMMSCs) have previously been shown to influence tumor progression and the development of chemoresistance. In the present study, we showed that when GFP labelled BMMSCs and RFP labelled HCC1806 cells are injected together in vivo, they create tumors which contain a new hybrid cell that has characteristics of both BMMSCs and HCC1806 cells. By labelling these cells prior to their injection, we were then able to isolate new hybrid cell from harvested tumors using FACS (DP-HCC1806:BMMSCs). Interestingly, when DP-HCC1806:BMMSCs were then injected into the mammary fat pad of NOD/SCID mice, they produced xenograft tumors which were smaller in size, and exhibited resistance to chemotherapy drugs (i.e. doxorubicin and 5-fluorouracil), when compared tumors from HCC1806 cells alone. This chemoresistance was shown to associated with an increased expression of tetraspanins (CD9, CD81) and drug resistance proteins (BCRP, MDR1). Subsequent siRNA-mediated knockdown of BMMSC-CD9 in DP-HCC1806:BMMSCs resulted in an attenuation of doxorubicin and 5-fluorouracil chemoresistance associated with decreased BCRP and serum cytokine expression (CCL5, CCR5, CXCR12). Our findings suggest that within the tumor microenvironment, CD9 is responsible for the crosstalk between BMMSCs and HCC1806 breast cancer cells (via CCL5, CCR5, and CXCR12) which contributes to chemoresistance. Hence, BMMSC-CD9 may serve as an important therapeutic target for the treatment of breast cancer.

A class of novel tubulin polymerization inhibitors exert effective anti-tumor activity via mitotic catastrophe.

In current work, a class of novel 4,5-dihydro-1H-pyrazole-1-carboxylate derivatives (E01-E28) were designed, synthesized and evaluated. Among them, the most potent compound E24 exhibited comparable activity against a panel of cancer cells (GI50 ranging 0.05-0.98 µM) and tubulin polymerization inhibition (IC50 = 1.49 µM) with reference drug CA-4(P) (GI50 ranging 0.019-0.32 µM, IC50 = 2.18 µM). The following assays indicated that compound E24 disturbed the dynamics of tubulin catastrophe and rescue, which triggered G2/M arrest, leading to ROS accumulation, cleavage of PARP and apoptosis. Molecular dynamics simulation validated that compound E24 could tightly bind into tubulin heterodimers with ß Lys 254 and ß Cys 241 of tubulin in the docking pose. Metabolic stability and pharmacokinetics parameters were also determined. The half time (t1/2) displayed species differences in three microsomes. The plasma elimination half-life (t1/2), peak plasma concentration (Cmax), mean retention time (MRT), the area under the curve (AUC0-∞) and distribution volume (Vz) of E24 after intravenous administration were 0.90 ± 0.22 h, 594.50 ± 97.23 ng/mL, 1.09 ± 0.22 h, 413.67 ± 105.64 ng/mL*h and 5.03 ± 1.82 L/kg, respectively. In HeLa-xenografts, compound E24 exhibited obvious antitumor efficacy via the suppression of tumor growth without weight loss of body or organ. In brief, compound E24 might be a hopeful candidate with excellent properties for oncotherapy as tubulin polymerization inhibitor.

Teroxirone suppresses growth and motility of human hepatocellular carcinoma cells.

AIMS: The prevalent human hepatocellular carcinoma (HCC) is a leading cause of global cancer-related mortality. The small molecular weight triepoxide derivative, 1,3,5-triazine-2,4,6(1H,3H,5H)-tri-one-1,3,5-tri-(oxiranylmethyl) (teroxirone), has been proved effective against the proliferation of lung cancer cells. The purpose is to further examine if teroxirone regulate growth and metastatic potential of HCC cells with aims at disclosing more of the reaction mechanisms. MAIN METHODS: Measurements of cell viability and flow cytometry were conducted to test sensitivities of teroxirone against HCC cells. The signaling pathway leading to apoptotic death was unraveled by Western blotting analysis. The metastatic progression was evaluated by cell-based phenotype assay that included migration, invasion, gelatin zymography and wound assay. The in vivo drug efficiency was done in immune-deficient mice with the established xenograft tumors. KEY FINDINGS: Teroxirone inhibited growth of HCC cells, but not hepatic cells. The drug induced apoptosis in HCC cells bearing mutant p53. Pretreatment of caspase-3 inhibitor restored cell viabilities by suppressing extrinsic pathway-mediated cell death. More experiments suggested that sub-apoptotic concentrations of teroxirone mitigated migration, invasion and wound healing of HCC cells. The drug reduced growth of the xenograft tumors as established in animal models by activating apoptotic death. SIGNIFICANCE: The findings asserted that teroxirone is an eligible addition to the existing options as an anticancer agent to eliminate HCC.

Analysis of CTCL cell lines reveals important differences between mycosis fungoides/Sézary syndrome vs. HTLV-1+ leukemic cell lines.

HTLV-1 is estimated to affect ~20 million people worldwide and in ~5% of carriers it produces Adult T-Cell Leukemia/Lymphoma (ATLL), which can often masquerade and present with classic erythematous pruritic patches and plaques that are typically seen in Mycosis Fungoides (MF) and Sézary Syndrome (SS), the most recognized variants of Cutaneous T-Cell Lymphomas (CTCL). For many years the role of HTLV-1 in the pathogenesis of MF/SS has been hotly debated. In this study we analyzed CTCL vs. HTLV-1+ leukemic cells. We performed G-banding/spectral karyotyping, extensive gene expression analysis, TP53 sequencing in the 11 patient-derived HTLV-1+ (MJ and Hut102) vs. HTLV-1- (Myla, Mac2a, PB2B, HH, H9, Hut78, SZ4, Sez4 and SeAx) CTCL cell lines. We further tested drug sensitivities to commonly used CTCL therapies and studied the ability of these cells to produce subcutaneous xenograft tumors in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Our work demonstrates that unlike classic advanced MF/SS cells that acquire many ongoing balanced and unbalanced chromosomal translocations, HTLV-1+ CTCL leukemia cells are diploid and exhibit only a minimal number of non-specific chromosomal alterations. Our results indicate that HTLV-1 virus is likely not involved in the pathogenesis of classic MF/SS since it drives a very different pathway of lymphomagenesis based on our findings in these cells. This study also provides for the first time a comprehensive characterization of the CTCL cells with respect to gene expression profiling, TP53 mutation status, ability to produce tumors in mice and response to commonly used therapies.

A potent indolylquinoline alleviates growth of human lung cancer cell tumorspheres.

To fight cancer at its roots by targeting cancer stem cells is a promising approach for therapy. Previously, an indolylquinoline derivative, 3-((7-ethyl-1H-indol-3-yl)-methyl)-2-methylquinoline (EMMQ), was reported effectively inhibiting the growth of lung cancer cells through impairment of cellular mitochondria functions. To address more on drug efficiency, the study further exploited if EMMQ can impede the propagation of tumorspheres stemmed from non-small cell lung cancer cells. EMMQ inhibited proliferation of spheroids in culture. In animal models, administration of the drug attenuated the spheroid tumorigenicity. The activated apoptosis alleviated growth of xenograft tumors in immune-deficient mice as established by the enriched tumorspheres. More evidence suggested that the reduced stemness of the spheroid tumors is attributed to apoptotic death. The findings supported that EMMQ is an eligible approach to eradicate the minor but tumorigenic lung cancer tumorspheres.

New polymer of lactic-co-glycolic acid-modified polyethylenimine for nucleic acid delivery.

AIM: To develop an improved delivery system for nucleic acids. MATERIALS & METHODS: We designed, synthesized and characterized a new polymer of lactic-co-glycolic acid-modified polyethylenimine (LGA-PEI). Functions of LGA-PEI polymer were determined. RESULTS: The new LGA-PEI polymer spontaneously formed nanoparticles (NPs) with DNA or RNA, and showed higher DNA or RNA loading efficiency, higher or comparable transfection efficacy, and lower cytotoxicity in several cell types including PANC-1, Jurkat and HEK293 cells, when compared with lipofectamine 2000, branched or linear PEI (25 kDa). In nude mouse models, LGA-PEI showed higher delivery efficiency of plasmid DNA or miRNA mimic into pancreatic and ovarian xenograft tumors. LGA-PEI/DNA NPs showed much lower toxicity than control PEI NPs in mouse models. CONCLUSION: The new LGA-PEI polymer is a safer and more effective system to deliver DNA or RNA than PEI.

CYB5D2 displays tumor suppression activities towards cervical cancer.

Cervical cancer is caused by infections with human papillomaviruses (HPV) and genetic alternations in the cervical epithelium. While the former is well studied, the latter remains unclear. We report here that CYB5D2/Neuferricin possesses tumor suppressing activity towards cervical tumorigenesis. Ectopic expression of CYB5D2 did not affect HeLa cell proliferation and the cell's ability to form xenograft tumors, but significantly inhibited HeLa cell invasion in vitro and the cell-produced lung metastasis in NOD/SCID mice. Knockdown of CYB5D2 enhanced HeLa cell invasion. Two mutations in CYB5D2, the substitutions of arginine (R) 7 with either proline (P) or glycine (G), were reported in colon cancer. Both CYB5D2(R7P) and CYB5D2(R7G) were incapable of inhibiting HeLa cell invasion. CYB5D2 binds heme, in which aspartate (D) 86 is required. While CYB5D2(D86G) is heme-binding defective, it inhibited HeLa cell invasion. On the other hand, CYB5D2(R7P) and CYB5D2(R7G) bound heme but did not inhibit HeLa cell invasion. Collectively, CYB5D2 inhibits HeLa cell invasion independently of its heme binding. Furthermore, immunohistochemistry examination of CYB5D2 expression in 20 normal cervical tissues and 40 cervical squamous cell carcinomas (SCC) revealed a CYB5D2 reduction in 87.5% (35/40) of SCC. Analysis of CYB5D2 gene expression and genomic alteration data available from Oncomeine™ detected significant reductions of CYB5D2 mRNA in 40 SCCs and CYB5D2 gene copy number in 107 SCCs. Collectively, we provide evidence that CYB5D2 is a candidate tumor suppressor of cervical tumorigenesis.

Pharmacokinetic Analysis of (64)Cu-ATSM Dynamic PET in Human Xenograft Tumors in Mice.

UNLABELLED: The aim of this study was to evaluate the feasibility to perform voxel-wise kinetic modeling on datasets obtained from tumor-bearing mice that underwent dynamic PET scans with (64)Cu-ATSM and extract useful physiological parameters. METHODS: Tumor-bearing mice underwent 90-min dynamic PET scans with (64)Cu-ATSM and CT scans with contrast. Irreversible and reversible two-tissue compartment models were fitted to time activity curves (TACs) obtained from whole tumor volumes and compared using the Akaike information criterion (AIC). Based on voxel-wise pharmacokinetic analysis, parametric maps of model rate constants k1, k3 and Ki were generated and compared to (64)Cu-ATSM uptake. RESULTS: Based on the AIC, an irreversible two-tissue compartment model was selected for voxel-wise pharmacokinetic analysis. Of the extracted parameters, k1 (~perfusion) showed a strong correlation with early tracer uptake (mean spearman R = 0.88) 5 min post injection (pi). Moreover, positive relationships were found between late tracer uptake (90 min pi) and both k3 and the net influx rate constant, Ki (mean spearman R = 0.56 and R = 0.86; respectively). CONCLUSION: This study shows the feasibility to extract relevant parameters from voxel-wise pharmacokinetic analysis to be used for preclinical validation of (64)Cu-ATSM as a hypoxia-specific PET tracer.

Models of neuroendocrine prostate cancer.

Prostate cancer remains the second leading cause of cancer death in men in the USA and most western countries. Prostatic acinar adenocarcinoma is the most commonly diagnosed form of prostate cancer. Small-cell neuroendocrine carcinoma is less frequently identified at the time of initial diagnosis, but this highly aggressive form of prostate cancer is increasingly observed in patients who have failed first- and second-line hormone therapy. Thus, developing and exploring models of neuroendocrine prostate cancer (NePC) are of increasing importance. This review examines the relevant xenograft tumor and genetically engineered mouse models of NePC, with the aim of addressing salient features and clinical relevance.