Ibulocydine Inhibits Migration and Invasion of TNBC Cells via MMP-9 Regulation.

Triple-negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancer types, indicating a poor survival prognosis with a more aggressive biology of metastasis to the lung and a short response duration to available therapies. Ibulocydine (IB) is a novel (cyclin-dependent kinase) CDK7/9 inhibitor prodrug displaying potent anti-cancer effects against various cancer cell types. We performed in vitro and in vivo experiments to determine whether IB inhibits metastasis and eventually overcomes the poor drug response in TNBC. The result showed that IB inhibited the growth of TNBC cells by inducing caspase-mediated apoptosis and blocking metastasis by reducing MMP-9 expression in vitro. Concurrently, in vivo experiments using the metastasis model showed that IB inhibited metastasis of MDA-MB-231-Luc cells to the lung. Collectively, these results demonstrate that IB inhibited the growth of TNBC cells and blocked metastasis by regulating MMP-9 expression, suggesting a novel therapeutic agent for metastatic TNBC.

Targeting phosphomevalonate kinase enhances radiosensitivity via ubiquitination of the replication protein A1 in lung cancer cells.

Radiotherapy (RT) plays an important role in localized lung cancer treatments. Although RT locally targets and controls malignant lesions, RT resistance prevents RT from being an effective treatment for lung cancer. In this study, we identified phosphomevalonate kinase (PMVK) as a novel radiosensitizing target and explored its underlying mechanism. We found that cell viability and survival fraction after RT were significantly decreased by PMVK knockdown in lung cancer cell lines. RT increased apoptosis, DNA damage, and G2/M phase arrest after PMVK knockdown. Also, after PMVK knockdown, radiosensitivity was increased by inhibiting the DNA repair pathway, homologous recombination, via downregulation of replication protein A1 (RPA1). RPA1 downregulation was induced through the ubiquitin-proteasome system. Moreover, a stable shRNA PMVK mouse xenograft model verified the radiosensitizing effects of PMVK in vivo. Furthermore, PMVK expression was increased in lung cancer tissues and significantly correlated with patient survival and recurrence. Our results demonstrate that PMVK knockdown enhances radiosensitivity through an impaired HR repair pathway by RPA1 ubiquitination in lung cancer, suggesting that PMVK knockdown may offer an effective therapeutic strategy to improve the therapeutic efficacy of RT.

ITC-6102RO, a novel B7-H3 antibody-drug conjugate, exhibits potent therapeutic effects against B7-H3 expressing solid tumors.

BACKGROUND: The B7-H3 protein, encoded by the CD276 gene, is a member of the B7 family of proteins and a transmembrane glycoprotein. It is highly expressed in various solid tumors, such as lung and breast cancer, and has been associated with limited expression in normal tissues and poor clinical outcomes across different malignancies. Additionally, B7-H3 plays a crucial role in anticancer immune responses. Antibody-drug conjugates (ADCs) are a promising therapeutic modality, utilizing antibodies targeting tumor antigens to selectively and effectively deliver potent cytotoxic agents to tumors. METHODS: In this study, we demonstrate the potential of a novel B7-H3-targeting ADC, ITC-6102RO, for B7-H3-targeted therapy. ITC-6102RO was developed and conjugated with dHBD, a soluble derivative of pyrrolobenzodiazepine (PBD), using Ortho Hydroxy-Protected Aryl Sulfate (OHPAS) linkers with high biostability. We assessed the cytotoxicity and internalization of ITC-6102RO in B7-H3 overexpressing cell lines in vitro and evaluated its anticancer efficacy and mode of action in B7-H3 overexpressing cell-derived and patient-derived xenograft models in vivo. RESULTS: ITC-6102RO inhibited cell viability in B7-H3-positive lung and breast cancer cell lines, inducing cell cycle arrest in the S phase, DNA damage, and apoptosis in vitro. The binding activity and selectivity of ITC-6102RO with B7-H3 were comparable to those of the unconjugated anti-B7-H3 antibody. Furthermore, ITC-6102RO proved effective in B7-H3-positive JIMT-1 subcutaneously xenografted mice and exhibited a potent antitumor effect on B7-H3-positive lung cancer patient-derived xenograft (PDX) models. The mode of action, including S phase arrest and DNA damage induced by dHBD, was confirmed in JIMT-1 tumor tissues. CONCLUSIONS: Our preclinical data indicate that ITC-6102RO is a promising therapeutic agent for B7-H3-targeted therapy. Moreover, we anticipate that OHPAS linkers will serve as a valuable platform for developing novel ADCs targeting a wide range of targets.

Bacterial indoleacetic acid-induced synthesis of colloidal Ag2O nanocrystals and their biological activities.

The biosynthesis and biological activity of colloidal Ag2O nanocrystals have not been well studied, although they have potential applications in many fields. For the first time, we developed a reducing agent free, cost-effective technique for Ag2O biosynthesis using Xanthomonas sp. P5. The optimal conditions for Ag2O synthesis were 50 °C, pH 8, and 2.5 mM AgNO3. Using these conditions the yield of Ag2O obtained at 10 h was about five times higher than that obtained at 12 h under unoptimized conditions. Ag2O was characterized by FESEM-EDS, TEM, dynamic light scattering, XRD, and UV-Visible spectroscopy. Indoleacetic acid produced by the strain P2 was involved in the synthesis of Ag2O. Ag2O exhibited a broad antimicrobial spectrum against several human pathogens. Furthermore, Ag2O exhibited 1,1-diphenyl-2-picrylhydrazyl (IC50 = 25.1 µg/ml) and 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonate (IC50 = 16.8 µg/ml) radical scavenging activities, and inhibited collagenase (IC50 = 27.9 mg/ml). Cytotoxicity of Ag2O was tested in fibroblast cells and found to be non-toxic, demonstrating biocompatibility.

Pyrrolidine dithiocarbamate reverses Bcl-xL-mediated apoptotic resistance to doxorubicin by inducing paraptosis.

Elevated Bcl-xL expression in cancer cells contributes to doxorubicin (DOX) resistance, leading to failure in chemotherapy. In addition, the clinical use of high-dose doxorubicin (DOX) in cancer therapy has been limited by issues with cardiotoxicity and hepatotoxicity. Here, we show that co-treatment with pyrrolidine dithiocarbamate (PDTC) attenuates DOX-induced apoptosis in Chang-L liver cells and human hepatocytes, but overcomes DOX resistance in Bcl-xL-overexpressing Chang-L cells and several hepatocellular carcinoma (HCC) cell lines with high Bcl-xL expression. Additionally, combined treatment with DOX and PDTC markedly retarded tumor growth in a Huh-7 HCC cell xenograft tumor model, compared to either mono-treatment. These results suggest that DOX/PDTC co-treatment may provide a safe and effective therapeutic strategy against malignant hepatoma cells with Bcl-xL-mediated apoptotic defects. We also found that induction of paraptosis, a cell death mode that is accompanied by dilation of the endoplasmic reticulum and mitochondria, is involved in this anti-cancer effect of DOX/PDTC. The intracellular glutathione levels were reduced in Bcl-xL-overexpressing Chang-L cells treated with DOX/PDTC, and DOX/PDTC-induced paraptosis was effectively blocked by pretreatment with thiol-antioxidants, but not by non-thiol antioxidants. Collectively, our results suggest that disruption of thiol homeostasis may critically contribute to DOX/PDTC-induced paraptosis in Bcl-xL-overexpressing cells.

Combination treatment with docetaxel and histone deacetylase inhibitors downregulates androgen receptor signaling in castration-resistant prostate cancer.

Backgrounds Since most patients with castration-resistant prostate cancer (CRPC) develop resistance to its standard therapy docetaxel, many studies have attempted to identify novel combination treatment to meet the large clinical unmet need. In this study, we examined whether histone deacetylase inhibitors (HDACIs) enhanced the effect of docetaxel on AR signaling in CRPC cells harboring AR and its splice variants. Methods HDACIs (vorinostat and CG200745) were tested for their ability to enhance the effects of docetaxel on cell viability and inhibition of AR signaling in CRPC 22Rv1 and VCaP cells by using CellTiter-Glo™ Luminescent cell viability assay, synergy index analysis and Western blotting. The nuclear localization of AR was examined via immunocytochemical staining in 22Rv1 cells and primary tumor cells from a patient with CRPC. Results Combination treatment with HDACIs (vorinostat or CG200745) and docetaxel synergistically inhibited the growth of 22Rv1 and VCaP cells. Consistently, the combination treatment decreased the levels of full-length AR (AR-FL), AR splice variants (AR-Vs), prostate-specific antigen (PSA), and anti-apoptotic Bcl-2 proteins more efficiently compared with docetaxel or vorinostat alone. Moreover, the combination treatment accelerated the acetylation and bundling of tubulin, which significantly inhibited the nuclear accumulation of AR in 22Rv1 cells. The cytoplasmic colocalization of AR-FL and AR-V7 with microtubule bundles increased after combination treatment in primary tumor cells from a patient with CRPC. Conclusions The results suggested that docetaxel, in combination with HDACIs, suppressed the expression and nuclear translocation of AR-FL and AR-Vs and showed synergistic anti-proliferative effect in CRPC cells. This combination therapy may be useful for the treatment of patients with CRPC.

Low immunogenic bio-nanocapsule based on hepatitis B virus escape mutants.

Bio-nanocapsules (BNCs) consisting of hepatitis B virus surface antigen (HBsAg) L proteins and phospholipids are used as efficient non-viral carriers for liver-specific delivery of genes and drugs. Considering the administration to HB vaccinees and HB patients, endogenous anti-HBsAg immunoglobulins (HBIGs) may reduce the delivery efficacy and prevent repetitive administration. Therefore, low immunogenic BNCs were generated by inserting two point mutations in the HBsAg L protein, which were found in HBV escape mutants. Escape mutant-type BNC (emBNC) showed 50% lower HBIG binding capacity than that of parental BNC (wtBNC). It induced HBIG production to a lesser extent than that associated with wtBNC in BALB/c mice. The emBNC could accumulate into human hepatocyte-derived tumor in mice pre-treated with HBIGs. The complex of emBNC and cationic liposomes could deliver plasmid DNA to HepG2 cells efficiently in the presence of HBIGs. Thus, emBNC could evade HBIG-neutralizing antibodies, expanding the clinical utility of BNC-based nanomedicine.

Improved biosynthesis of silver nanoparticles using keratinase from Stenotrophomonas maltophilia R13: reaction optimization, structural characterization, and biomedical activity.

In the present study, keratinase from Stenotrophomonas maltophilia R13 was used for the first time as a reducing agent for the eco-friendly synthesis of AgNPs. The keratinase produced by strain R13 was responsible for the reduction of silver ions and the subsequent formation of AgNPs. Maximum AgNP synthesis was achieved using 2 mM AgNO3 at pH 9 and 40 °C. Electron microscopy and dynamic light scattering analysis showed AgNPs were spherical and of average diameter ~ 8.4 nm. X-ray diffraction revealed that AgNPs were crystalline. FTIR indicated AgNPs were stabilized by proteins present in the crude enzyme solution of strain R13. AgNPs exhibited a broad antimicrobial spectrum against several pathogenic microorganisms, and the antimicrobial mechanism appeared to involve structural deformation of cells resulting in membrane leakage and subsequent lysis. AgNPs also displayed 1,1-diphenyl-2-picrylhydrazyl (IC50 = 0.0112 mg/ml), 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonate radical scavenging (IC50 = 0.0243 mg/ml), and anti-collagenase (IC50 = 23.5 mg/ml) activities.

Establishment and characterization of 6 novel patient-derived primary pancreatic ductal adenocarcinoma cell lines from Korean pancreatic cancer patients.

BACKGROUND: Pancreatic ductal adenocarcinomas are among the most malignant neoplasms and have very poor prognosis. Our understanding of various cancers has recently improved the survival of patients with cancer, except for pancreatic cancers. Establishment of primary cancer cell lines of pancreatic ductal adenocarcinomas will be useful for understanding the molecular mechanisms of this disease. METHODS: Eighty-one surgically resected pancreatic ductal adenocarcinomas were collected. Six novel pancreatic cancer cell lines, AMCPAC01-06, were established and histogenetic characteristics were compared with their matched tissues. The clinicopathologic and molecular characteristics of the cell lines were investigated by KRAS and TP53 sequencing or SMAD4 and p53 immunohistochemistry. Xenografts using AMCPAC cell lines were established. RESULTS: From the 81 pancreatic ductal adenocarcinomas, six (7.4% success rate) patient-derived primary cell lines were established. The six AMCPAC cell lines showed various morphologies and exhibited a wide range of doubling times. AMCPAC cell lines contained mutant KRAS in codons 12, 13, or 61 and TP53 in exon 5 as well as showed aberrant p53 (5 overexpression and 1 total loss) or DPC4 (all 6 intact) expression. AMCPAC cell lines demonstrated homology for the KRAS mutation and p53 expression compared with matched primary cancer tissues, but showed heterogeneous DPC4 expression patterns. CONCLUSIONS: The novel AMCPAC01-06 cell lines established in this study may contribute to the understanding of pancreatic ductal adenocarcinomas. Trial registration Retrospectively registered.

Application of peptide displaying phage as a novel diagnostic probe for human lung adenocarcinoma.

Despite the increasing lung cancer-associated death rate, its therapy has been constrained by impasse of early diagnosis. To apply non-invasive imaging for potential cancer diagnosis system, we screened human lung adenocarcinoma-specific peptides using the phage display technique. For in vivo phage-displayed peptide screening, M13 phage library displaying 2.9 × 10(9) random peptides was injected through tail vein to lung adenocarcinoma cell-derived xenograft mouse model. Through four rounds of biopanning, a specific peptide sequence (CAKATCPAC) was screened out with the highest frequency and was named as Pep-1, and it was analyzed for its targeting ability as an imaging probe by in vitro competitive assay to test its cell-binding ability, immunohistochemical detection in the tumor tissue, and in vivo NIR fluorescent optical imaging. The specificity of Pep-1 toward lung cancer was ensured by in vivo imaging using xenograft animals of various cancer types. The results suggest that Pep-1 is a promising diagnostic lead molecule for rapid and accurate detection of human lung adenocarcinoma. In addition, it was found that the targeting ability was much enhanced by ionizing radiation in both cell-derived and patient-derived lung adenocarcinoma xenografts, suggesting the possibility of applying Pep-1 for prognostic diagnosis after radiotherapy. Taken together, this study suggests that Pep-1 possesses a specific-targeting ability for human lung adenocarcinoma and that this peptide could be directly used as a clinically applicable imaging probe.

Novel peptides functionally targeting in vivo human lung cancer discovered by in vivo peptide displayed phage screening.

Discovery of the cancer-specific peptidic ligands have been emphasized for active targeting drug delivery system and non-invasive imaging. For the discovery of useful and applicable peptidic ligands, in vivo peptide-displayed phage screening has been performed in this study using a xenograft mouse model as a mimic microenvironment to tumor. To seek human lung cancer-specific peptides, M13 phage library displaying 2.9 × 10(9) random peptides was intravenously injected into mouse model bearing A549-derived xenograft tumor through the tail vein. Then the phages emerged from a course of four rounds of biopanning in the xenograft tumor tissue. Novel peptides were categorized into four groups according to a sequence-homology phylogenicity, and in vivo tumor-targeting capacity of these peptides was validated by whole body imaging with Cy5.5-labeled phages in various cancer types. The result revealed that novel peptides accumulated only in adenocarcinoma lung cancer cell-derived xenograft tissue. For further confirmation of the specific targeting ability, in vitro cell-binding assay and immunohistochemistry in vivo tumor tissue were performed with a selected peptide. The peptide was found to bind intensely to lung cancer cells both in vitro and in vivo, which was efficiently compromised with unlabeled phages in an in vitro competition assay. In conclusion, the peptides specifically targeting human lung cancer were discovered in this study, which is warranted to provide substantive feasibilities for drug delivery and imaging in terms of a novel targeted therapeutics and diagnostics.

Determination of Optimal Antigen Yield and Virus Inactivation Conditions for the Production of the Candidate Foot-and-Mouth Disease Recombinant Vaccine Strain Asia1 Shamir-R in a Bioreactor.

Since the foot-and-mouth disease (FMD) outbreak in South Korea in 2010-2011, vaccination policies utilizing inactivated FMD vaccines composed of types O and A have been implemented nationwide. However, because type Asia1 occurred in North Korea in 2007 and intermittently in neighboring countries, the risk of type Asia1 introduction cannot be ruled out. This study evaluated the antigen yield and viral inactivation kinetics of the recombinant Asia1 Shamir vaccine strain (Asia1 Shamir-R). When Asia1 Shamir-R was proliferated in shaking flasks (1 L), a 2 L bioreactor (1 L), and a wave bioreactor (25 L), the antigen yields were 7.5 µg/mL, 5.2 µg/mL, and 3.8 µg/mL, respectively. The optimal FMDV inactivation conditions were 2 mM BEI at 26 °C and 1.0 mM BEI at 37 °C. There was no antigen loss due to BEI treatment, and only a decrease in antigen levels was observed during storage. The sera from pigs immunized with antigen derived from a bioreactor exhibited a neutralizing antibody titer of approximately 1/1000 against Asia1 Shamir and Asia1/MOG/05 viruses; therefore, Asia1 Shamir-R is expected to provide sufficient protection against both viruses. If an FMD vaccine production facility is established, this Asia1 Shamir-R can be employed for domestic antigen banks in South Korea.

Evaluation of Foot-and-Mouth Disease (FMD) Virus Asia1 Genotype-V as an FMD Vaccine Candidate: Study on Vaccine Antigen Production Yield and Inactivation Kinetics.

South Korea has experienced outbreaks of foot-and-mouth disease (FMD) of serotypes O and A, leading to nationwide vaccination with a bivalent vaccine. Since the FMD virus (FMDV) Asia1 group-V genotype occurred in North Korea in 2007, an Asia1/MOG/05 vaccine strain belonging to the Asia1 group-V genotype was developed using a genetic recombination method (Asia1/MOG/05-R). This study aimed to evaluate the antigen productivity and viral inactivation kinetics of Asia1/MOG/05-R to assess its commercial viability. The antigen yield of Asia1/MOG/05-R produced in flasks and bioreactors was approximately 4.0 µg/mL. Binary ethylenimine (BEI) inactivation kinetics of Asia1/MOG/05-R showed that 2 mM and 1.0 mM BEI treatment at 26 °C and 37 °C, respectively, resulted in a virus titer <10-7 TCID50/mL within 24 h, meeting the inactivation kinetics criteria. During incubation at 26 °C and 37 °C, 10% antigen loss occurred, but not due to BEI treatment. When pigs were inoculated twice with the Asia1/MOG/05-R antigen, the virus neutralization titer increased to approximately 1:1000; therefore, it can sufficiently protect against Asia1/MOG/05-R and Asia1 Shamir viruses. The Asia1/MOG/05-R will be useful as a vaccine strain for domestic antigen banks.

Akt enhances the vulnerability of cancer cells to VCP/p97 inhibition-mediated paraptosis.

Valosin-containing protein (VCP)/p97, an AAA+ ATPase critical for maintaining proteostasis, emerges as a promising target for cancer therapy. This study reveals that targeting VCP selectively eliminates breast cancer cells while sparing non-transformed cells by inducing paraptosis, a non-apoptotic cell death mechanism characterized by endoplasmic reticulum and mitochondria dilation. Intriguingly, oncogenic HRas sensitizes non-transformed cells to VCP inhibition-mediated paraptosis. The susceptibility of cancer cells to VCP inhibition is attributed to the non-attenuation and recovery of protein synthesis under proteotoxic stress. Mechanistically, mTORC2/Akt activation and eIF3d-dependent translation contribute to translational rebound and amplification of proteotoxic stress. Furthermore, the ATF4/DDIT4 axis augments VCP inhibition-mediated paraptosis by activating Akt. Given that hyperactive Akt counteracts chemotherapeutic-induced apoptosis, VCP inhibition presents a promising therapeutic avenue to exploit Akt-associated vulnerabilities in cancer cells by triggering paraptosis while safeguarding normal cells.

Application of deep learning technology for temporal analysis of videofluoroscopic swallowing studies.

Temporal parameters during swallowing are analyzed for objective and quantitative evaluation of videofluoroscopic swallowing studies (VFSS). Manual analysis by clinicians is time-consuming, complicated and prone to human error during interpretation; therefore, automated analysis using deep learning has been attempted. We aimed to develop a model for the automatic measurement of various temporal parameters of swallowing using deep learning. Overall, 547 VFSS video clips were included. Seven temporal parameters were manually measured by two physiatrists as ground-truth data: oral phase duration, pharyngeal delay time, pharyngeal response time, pharyngeal transit time, laryngeal vestibule closure reaction time, laryngeal vestibule closure duration, and upper esophageal sphincter opening duration. ResNet3D was selected as the base model for the deep learning of temporal parameters. The performances of ResNet3D variants were compared with those of the VGG and I3D models used previously. The average accuracy of the proposed ResNet3D variants was from 0.901 to 0.981. The F1 scores and average precision were 0.794 to 0.941 and 0.714 to 0.899, respectively. Compared to the VGG and I3D models, our model achieved the best results in terms of accuracy, F1 score, and average precision values. Through the clinical application of this automatic model, temporal analysis of VFSS will be easier and more accurate.

Orthotopic model of pancreatic cancer using CD34+ humanized mice and generation of tumor organoids from humanized tumors.

In pancreatic cancer (PC) as intractable solid cancer, current research is focused mainly on targeted immunotherapies such as antibodies and immune cell modulators. To identify promising immune-oncological agents, animal models that recapitulate the essential features of human immune status are essential. To this end, we constructed an orthotopic xenograft model using CD34+ human hematopoietic stem cell-based humanized NOD scid gamma mouse (NSG) mice injected with luciferase-expressing PC cell lines AsPC1 and BxPC3. The growth of orthotopic tumors was monitored using noninvasive multimodal imaging, while the subtype profiles of human immune cells in blood and tumor tissues were determined by flow cytometry and immunohistopathology. In addition, the correlations of blood and tumor-infiltrating immune cell count with tumor extracellular matrix density were calculated using Spearman's test. Tumor-derived cell lines and tumor organoids with continuous passage capacity in vitro were isolated from orthotopic tumors. It was further confirmed that these tumor-derived cells and organoids have reduced PD-L1 expression and are suitable for testing the efficacy of specific targeted immunotherapeutic agents. These animal and culture models could facilitate the development and validation of immunotherapeutic agents for intractable solid cancers including PC.

NCK-associated protein 1 regulates metastasis and is a novel prognostic marker for colorectal cancer.

Metastatic colorectal cancer (CRC) remains a substantial problem for mortality and requires screening and early detection efforts to increase survival. Epithelial-mesenchymal transition (EMT) and circulation of tumor cells in the blood play important roles in metastasis. To identify a novel target for metastasis of CRC, we conducted a gene microarray analysis using extracted RNA from the blood of preclinical models. We found that NCK-associated protein 1 (NCKAP1) was significantly increased in the blood RNA of patient-derived xenograft (PDX) models of colon cancer. In the NCKAP1 gene knockdown-induced human colon cancer cell lines HCT116 and HT29, there was a reduced wound healing area and significant inhibition of migration and invasion. As the result of marker screening for cytoskeleton and cellular interactions, CRC treated with siRNA of NCKAP1 exhibited significant induction of CDH1 and phalloidin expression, which indicates enhanced adherent cell junctions and cytoskeleton. In HCT116 cells with a mesenchymal state induced by TGFß1, metastasis was inhibited by NCKAP1 gene knockdown through the inhibition of migration, and there was increased CTNNB1 expression and decreased FN expression. We established metastasis models for colon cancer to liver transition by intrasplenic injection shRNA of NCKAP1-transfected HCT116 cells or by implanting tumor tissue generated with the cells on cecal pouch. In metastasis xenograft models, tumor growth and liver metastasis were markedly reduced. Taken together, these data demonstrate that NCKAP1 is a novel gene regulating EMT that can contribute to developing a diagnostic marker for the progression of metastasis and new therapeutics for metastatic CRC treatment.

Carnitine sensitizes TRAIL-resistant cancer cells to TRAIL-induced apoptotic cell death through the up-regulation of Bax.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family with apoptosis-inducing activity. Given that TRAIL selectively induces cell death in various tumors but has little or no toxicity to normal cells, TRAIL agonists have been considered as promising anti-cancer therapeutic agents. However, the resistance of many primary tumors and cancer cells to TRAIL poses a challenge. In our present study, we found that carnitine, a metabolite that transfers long-chain fatty acids into mitochondria for beta-oxidation and modulates protein kinase C activity, sensitizes TRAIL-resistant cancer cells to TRAIL. Combination of carnitine and TRAIL was found to synergistically induce apoptotic cell death through caspase activation, which was blocked by a pan caspase inhibitor, but not by an inhibitor of autophagy or an inhibitor of necrosis. The combination of carnitine and TRAIL reversed the resistance to TRAIL in lung cancer cells, colon carcinoma cells, and breast carcinoma cells. We further demonstrate that carnitine, either alone or in combination with TRAIL, enhances the expression of the pro-apoptotic Bcl-2 family protein, Bcl-2-associated X protein (Bax). The down-regulation of Bax expression by small interfering RNA reduced caspase activation when cells were treated with TRAIL, and experiments with cells from Bax knockout mice confirmed this result. Taken together, our current results suggest that carnitine can reverse the resistance of cancer cells to TRAIL by up-regulating Bax expression. Thus, a combined delivery of carnitine and TRAIL may represent a new therapeutic strategy to treat TRAIL-resistant cancer cells.

Novel histone deacetylase inhibitor CG200745 induces clonogenic cell death by modulating acetylation of p53 in cancer cells.

Histone deacetylase (HDAC) plays an important role in cancer onset and progression. Therefore, inhibition of HDAC offers potential as an effective cancer treatment regimen. CG200745, (E)-N(1)-(3-(dimethylamino)propyl)-N(8)-hydroxy-2-((naphthalene-1-loxy)methyl)oct-2-enediamide, is a novel HDAC inhibitor presently undergoing a phase I clinical trial. Enhancement of p53 acetylation by HDAC inhibitors induces cell cycle arrest, differentiation, and apoptosis in cancer cells. The purpose of the present study was to investigate the role of p53 acetylation in the cancer cell death caused by CG200745. CG200745-induced clonogenic cell death was 2-fold greater in RKO cells expressing wild-type p53 than in p53-deficient RC10.1 cells. CG200745 treatment was also cytotoxic to PC-3 human prostate cancer cells, which express wild-type p53. CG200745 increased acetylation of p53 lysine residues K320, K373, and K382. CG200745 induced the accumulation of p53, promoted p53-dependent transactivation, and enhanced the expression of MDM2 and p21(Waf1/Cip1) proteins, which are encoded by p53 target genes. An examination of CG200745 effects on p53 acetylation using cells transfected with various p53 mutants showed that cells expressing p53 K382R mutants were significantly resistant to CG200745-induced clonogenic cell death compared with wild-type p53 cells. Moreover, p53 transactivation in response to CG200745 was suppressed in all cells carrying mutant forms of p53, especially K382R. Taken together, these results suggest that acetylation of p53 at K382 plays an important role in CG200745-induced p53 transactivation and clonogenic cell death.

PAUF as a Target for Treatment of High PAUF-Expressing Ovarian Cancer.

Pancreatic adenocarcinoma up-regulated factor (PAUF) plays an important role in tumor growth, metastasis, and immune evasion in the pancreatic tumor microenvironment, and recent studies suggest an association between PAUF expression and poor prognosis in ovarian cancer patients. The current study aimed 1) to characterize the potential tumor-promoting role of PAUF in ovarian cancer, using in vitro and in vivo models, including a PAUF-knockout OVCAR-5 cell line, and 2) to explore the potential therapeutic effects of an anti-PAUF antibody for ovarian cancer. Recombinant PAUF significantly increased tumor metastatic capacity (migration, invasion, and adhesion) in all the ovarian cancer cell lines tested, except for the OVCAR-5 cell line which expresses PAUF at a much higher level than the other cells. PAUF-knockout in the OVCAR-5 cell line led to apparently delayed tumor growth in vitro and in vivo. Furthermore, the administration of an anti-PAUF antibody exhibited notable sensitizing and synchronizing effects on docetaxel in mice bearing the OVCAR-5 xenograft tumors. Taken together, this study shows that the expression level of PAUF is an independent factor determining malignant behaviors of ovarian cancer and, for the first time, it suggests that PAUF may be a promising therapeutic target for high PAUF-expressing ovarian cancer.