Molecular Pathways Governing the Termination of Liver Regeneration.

The liver has the unique capacity to regenerate, and up to 70% of the liver can be removed without detrimental consequences to the organism. Liver regeneration is a complex process involving multiple signaling networks and organs. Liver regeneration proceeds through three phases: the initiation phase, the growth phase, and the termination phase. Termination of liver regeneration occurs when the liver reaches a liver-to-body weight that is required for homeostasis, the so-called "hepatostat." The initiation and growth phases have been the subject of many studies. The molecular pathways that govern the termination phase, however, remain to be fully elucidated. This review summarizes the pathways and molecules that signal the cessation of liver regrowth after partial hepatectomy and answers the question, "What factors drive the hepatostat?" SIGNIFICANCE STATEMENT: Unraveling the pathways underlying the cessation of liver regeneration enables the identification of druggable targets that will allow us to gain pharmacological control over liver regeneration. For these purposes, it would be useful to understand why the regenerative capacity of the liver is hampered under certain pathological circumstances so as to artificially modulate the regenerative processes (e.g., by blocking the cessation pathways) to improve clinical outcomes and safeguard the patient's life.

Comparative analysis of whole cell-derived vesicular delivery systems for photodynamic therapy of extrahepatic cholangiocarcinoma.

This first-in-its-class proof-of-concept study explored the use of bionanovesicles for the delivery of photosensitizer into cultured cholangiocarcinoma cells and subsequent treatment by photodynamic therapy (PDT). Two types of bionanovesicles were prepared: cellular vesicles (CVs) were fabricated by sonication-mediated nanosizing of cholangiocarcinoma (TFK-1) cells, whereas cell membrane vesicles (CMVs) were produced by TFK-1 cell and organelle membrane isolation and subsequent nanovesicularization by sonication. The bionanovesicles were loaded with zinc phthalocyanine (ZnPC). The CVs and CMVs were characterized (size, polydispersity index, zeta potential, stability, ZnPC encapsulation efficiency, spectral properties) and assayed for tumor (TFK-1) cell association and uptake (flow cytometry, confocal microscopy), intracellular ZnPC distribution (confocal microscopy), dark toxicity (MTS assay), and PDT efficacy (MTS assay). The mean ±â€¯SD diameter, polydispersity index, and zeta potential were 134 ±â€¯1 nm, -16.1 ±â€¯0.9, and 0.220 ±â€¯0.013, respectively, for CVs and 172 ±â€¯3 nm, -16.4 ±â€¯1.1, and 0.167 ±â€¯0.022, respectively, for CMVs. Cold storage for 1 wk and incorporation of ZnPC increased bionanovesicular diameter slightly but size remained within the recommended range for in vivo application (136-220 nm). ZnPC was incorporated into CVs and CMVs at an optimal photosensitizer:lipid molar ratio of 0.006 and 0.01, respectively. Both bionanovesicles were avidly taken up by TFK-1 cells, resulting in homogenous intracellular ZnPC dispersion. Photosensitization of TFK-1 cells did not cause dark toxicity, while illumination at 671 nm (35.3 J/cm2) produced LC50 values of 1.11 µM (CVs) and 0.51 µM (CMVs) at 24 h post-PDT, which is superior to most LC50 values generated in tumor cells photosensitized with liposomal ZnPC. In conclusion, CVs and CMVs constitute a potent photosensitizer platform with no inherent cytotoxicity and high PDT efficacy in vitro.

Nanobody-mediated targeting of zinc phthalocyanine with polymer micelles as nanocarriers.

Photodynamic therapy (PDT) is a suitable alternative to currently employed cancer treatments. However, the hydrophobicity of most photosensitizers (e.g., zinc phthalocyanine (ZnPC)) leads to their aggregation in blood. Moreover, non-specific accumulation in skin and low clearance rate of ZnPC leads to long-lasting skin photosensitization, forcing patients with a short life expectancy to remain indoors. Consequently, the clinical implementation of these photosensitizers is limited. Here, benzyl-poly(ε-caprolactone)-b-poly(ethylene glycol) micelles encapsulating ZnPC (ZnPC-M) were investigated to increase the solubility of ZnPC and its specificity towards cancers cells. Asymmetric flow field-flow fractionation was used to characterize micelles with different ZnPC-to-polymer ratios and their stability in human plasma. The ZnPC-M with the lowest payload (0.2 and 0.4% ZnPC w/w) were the most stable in plasma, exhibiting minimal ZnPC transfer to lipoproteins, and induced the highest phototoxicity in three cancer cell lines. Nanobodies (Nbs) with binding specificity towards hepatocyte growth factor receptor (MET) or epidermal growth factor receptor (EGFR) were conjugated to ZnPC-M to facilitate cell targeting and internalization. MET- and EGFR-targeting micelles enhanced the association and the phototoxicity in cells expressing the target receptor. Altogether, these results indicate that ZnPC-M decorated with Nbs targeting overexpressed proteins on cancer cells may provide a better alternative to currently approved formulations.

Prognostic and predictive value of human equilibrative nucleoside transporter 1 (hENT1) in extrahepatic cholangiocarcinoma: a translational study.

Introduction: Effective (neo) adjuvant chemotherapy for cholangiocarcinoma is lacking due to chemoresistance and the absence of predictive biomarkers. Human equilibrative nucleoside transporter 1 (hENT1) has been described as a potential prognostic and predictive biomarker. In this study, the potential of rabbit-derived (SP120) and murine-derived (10D7G2) antibodies to detect hENT1 expression was compared in tissue samples of patients with extrahepatic cholangiocarcinoma (ECC), and the predictive value of hENT1 was investigated in three ECC cell lines. Methods: Tissues of 71 chemonaïve patients with histological confirmation of ECC were selected and stained with SP120 or 10D7G2 to assess the inter-observer variability for both antibodies and the correlation with overall survival. Concomitantly, gemcitabine sensitivity after hENT1 knockdown was assessed in the ECC cell lines EGI-1, TFK-1, and SK-ChA-1 using sulforhodamine B assays. Results: Scoring immunohistochemistry for hENT1 expression with the use of SP120 antibody resulted in the highest interobserver agreement but did not show a prognostic role of hENT1. However, 10D7G2 showed a prognostic role for hENT1, and a potential predictive role for gemcitabine sensitivity in hENT1 in SK-ChA-1 and TFK-1 cells was found. Discussion: These findings prompt further studies for both preclinical validation of the role of hENT1 and histochemical standardization in cholangiocarcinoma patients treated with gemcitabine-based chemotherapy.

S1PR1 regulates ovarian cancer cell senescence through the PDK1-LATS1/2-YAP pathway.

Cell senescence deters the activation of various oncogenes. Induction of senescence is, therefore, a potentially effective strategy to interfere with vital processes in tumor cells. Sphingosine-1-phosphate receptor 1 (S1PR1) has been implicated in various cancer types, including ovarian cancer. The mechanism by which S1PR1 regulates ovarian cancer cell senescence is currently elusive. In this study, we demonstrate that S1PR1 was highly expressed in human ovarian cancer tissues and cell lines. S1PR1 deletion inhibited the proliferation and migration of ovarian cancer cells. S1PR1 deletion promoted ovarian cancer cell senescence and sensitized ovarian cancer cells to cisplatin chemotherapy. Exposure of ovarian cancer cells to sphingosine-1-phosphate (S1P) increased the expression of 3-phosphatidylinositol-dependent protein kinase 1 (PDK1), decreased the expression of large tumor suppressor 1/2 (LATS1/2), and induced phosphorylation of Yes-associated protein (p-YAP). Opposite results were obtained in S1PR1 knockout cells following pharmacological inhibition. After silencing LATS1/2 in S1PR1-deficient ovarian cancer cells, senescence was suppressed and S1PR1 expression was increased concomitantly with YAP expression. Transcriptional regulation of S1PR1 by YAP was confirmed by chromatin immunoprecipitation. Accordingly, the S1PR1-PDK1-LATS1/2-YAP pathway regulates ovarian cancer cell senescence and does so through a YAP-mediated feedback loop. S1PR1 constitutes a druggable target for the induction of senescence in ovarian cancer cells. Pharmacological intervention in the S1PR1-PDK1-LATS1/2-YAP signaling axis may augment the efficacy of standard chemotherapy.

Infection prevention and control in Indonesian hospitals: identification of strengths, gaps, and challenges.

BACKGROUND: Infection prevention and control (IPC) in hospitals is key to safe patient care. There is currently no data regarding the implementation of IPC in hospitals in Indonesia. The aim of this study was to assess the existing IPC level in a nationwide survey, using the World Health Organization (WHO) IPC assessment framework tool (IPCAF), and to identify strengths, gaps, and challenges. METHODS: A cross-sectional study was conducted from July to November 2021. Of all general hospitals in Indonesia, 20% (N = 475) were selected using stratified random sampling based on class (A, B, C and D; class D with a maximum of 50 beds and class A with ≥ 250 beds) and region. The IPCAF was translated into Indonesian and tested in four hospitals. Questions were added regarding challenges in the implementation of IPC. Quantitative IPCAF scores are reported as median (minimum-maximum). IPC levels were calculated according to WHO tools. RESULTS: In total, 355 hospitals (74.7%) participated in this study. The overall median IPCAF score was 620.0 (535.0-687.5). The level of IPC was mostly assessed as advanced (56.9% of hospitals), followed by intermediate (35.8%), basic (7.0%) and inadequate (0.3%). In the eastern region of the country, the majority of hospitals scored intermediate level. Of the eight core components, the one with the highest score was IPC guidelines. Almost all hospitals had guidelines on the most important topics, including hand hygiene. Core components with the lowest score were surveillance of healthcare-associated infections (HAIs), education and training, and multimodal strategies. Although > 90% of hospitals indicated that surveillance of HAIs was performed, 57.2% reported no availability of adequate microbiology laboratory capacity to support HAIs surveillance. The most frequently reported challenges in the implementation of IPC were communication with the management of the hospitals, followed by the unavailability of antimicrobial susceptibility testing results and insufficient staffing of full-time IPC nurses. CONCLUSION: The IPC level in the majority of Indonesian hospitals was assessed as advanced, but there was no even distribution over the country. The IPCAF in combination with interviews identified several priority areas for interventions to improve IPC in Indonesian hospitals.

No difference in treatment outcome between patients with nodal versus extranodal diffuse large B-cell lymphoma.

Background and Aim: Diffuse large B-cell lymphoma (DLBCL) has been classified using various parameters, including the site of origin. Studies have reported conflicting outcomes when DLBLC patients were stratified according to the site of origin. This study aimed to investigate the response rate and survival outcomes in nodal versus extranodal DLBCL and compare the results to a region-matched study covering the 1988 - 2005 period. Methods: A single-center retrospective cohort study was conducted on all patients diagnosed with DLBCL and treated in a tertiary care hospital in Pakistan during 2014 - 2019. We calculated the mean and median for continuous variables and frequency and percentages for all categorical variables. Progression-free survival (PFS) and overall survival (OS) were calculated using Kaplan-Meier survival curves. A Cox proportional hazards model was used to determine the hazard ratio (HR) for OS. Results: Of the 118 patients, 49 patients (41.5%) had nodal disease and 69 patients (58.5%) were diagnosed with extranodal DLBCL. The majority of patients in the nodal and extranodal cohorts presented with Stages III and IV disease (73.4% and 62.3%, respectively). A complete response to (immuno) chemotherapy was achieved in 71.4% of nodal DLBCL patients and 65.2% of extranodal DLBCL patients. The 5-year PFS and median PFS in the entire cohort were 0.8% and 17 m, respectively. The PFS and median PFS in the nodal and extranodal DLBCL cohort were 0% and 1.4%, respectively, and 15 m and 19 m, respectively. The 5-year OS and median OS in the entire cohort were 16.1% and 19 m, respectively. The OS and median OS in the nodal and extranodal DLBCL cohort were 8.2% and 21.7%, respectively, and 19 m and 21 m, respectively. Multivariable linear regression revealed that the ABC phenotype (nodal, HR = 1.37, 95% CI = 1.37 - 3.20; extranodal, HR = 1.65, 95% CI = 1.46 - 3.17; GBC as reference) and double and triple hit DLBCL (nodal, HR = 1.29, 95% CI = 1.19 - 2.81; extranodal, HR = 1.87, 95% CI = 1.28 - 2.43; and non-expressors as reference) are independent negative predictors of OS. Conclusions: DLBCL incidence in the Karachi region has remained comparable but patient composition in the extranodal DLBCL cohort has shifted to predominantly advanced stage. Nodal and extranodal DLBCL were associated with similar PFS and OS profiles and first- and second-line treatment responses. Cell of origin and antigen expression status was independent negative predictors of OS, disfavoring the ABC phenotype and lesions with c-MYC and BCL2 and/or BCL6 overexpression. Relevance for Patients: DLBCL is an aggressive type of non-Hodgkin's lymphoma, however; patients respond well to standard systemic chemotherapy. Extranodal type of DLBCL patients tend to have more residual disease after first-line systemic chemotherapy, but physicians should keep in mind that the subsequent line treatment mitigates its negative impact on survival.

The Impact of Nanobody Density on the Targeting Efficiency of PEGylated Liposomes.

Nanoparticles (NPs) are commonly modified with tumor-targeting moieties that recognize proteins overexpressed on the extracellular membrane to increase their specific interaction with target cells. Nanobodies (Nbs), the variable domain of heavy chain-only antibodies, are a robust targeting ligand due to their small size, superior stability, and strong binding affinity. For the clinical translation of targeted Nb-NPs, it is essential to understand how the number of Nbs per NP impacts the receptor recognition on cells. To study this, Nbs targeting the hepatocyte growth factor receptor (MET-Nbs) were conjugated to PEGylated liposomes at a density from 20 to 800 per liposome and their targeting efficiency was evaluated in vitro. MET-targeted liposomes (MET-TLs) associated more profoundly with MET-expressing cells than non-targeted liposomes (NTLs). MET-TLs with approximately 150-300 Nbs per liposome exhibited the highest association and specificity towards MET-expressing cells and retained their targeting capacity when pre-incubated with proteins from different sources. Furthermore, a MET-Nb density above 300 Nbs per liposome increased the interaction of MET-TLs with phagocytic cells by 2-fold in ex vivo human blood compared to NTLs. Overall, this study demonstrates that adjusting the MET-Nb density can increase the specificity of NPs towards their intended cellular target and reduce NP interaction with phagocytic cells.

A tumor microenvironment-responsive micelle co-delivered radiosensitizer Dbait and doxorubicin for the collaborative chemo-radiotherapy of glioblastoma.

Glioblastoma is rather recalcitrant to existing therapies and effective interventions are needed. Here we report a novel microenvironment-responsive micellar system (ch-K5(s-s)R8-An) for the co-delivery of the radiosensitizer Dbait and the chemotherapeutic doxorubicin (DOX) to glioblastoma. Accordingly, the ch-K5(s-s)R8-An/(Dbait-DOX) micelles plus radiotherapy (RT) treatment resulted in a high degree of apoptosis and DNA damage, which significantly reduced cell viability and proliferation capacity of U251 cells to 64.0% and 16.3%, respectively. The angiopep-2-modified micelles exhibited substantial accumulation in brain-localized U251 glioblastoma xenografts in mice compared to angiopep-2-lacking micelles. The ch-K5(s-s)R8-An/(Dbait-DOX) + RT treatment group exhibited the smallest tumor size and most profound tumor tissue injury in orthotopic U251 tumors, leading to an increase in median survival time of U251 tumor-bearing mice from 26 days to 56 days. The ch-K5(s-s)R8-An/(Dbait-DOX) micelles can be targeted to brain-localized U251 tumor xenografts and sensitize the tumor to chemotherapy and radiotherapy, thereby overcoming the inherent therapeutic challenges associated with malignant glioblastoma.

Utility of Intravenous Curcumin Nanodelivery Systems for Improving In Vivo Pharmacokinetics and Anticancer Pharmacodynamics.

Curcumin nanoformulations for intravenous injection have been developed to offset poor absorption, biotransformation, degradation, and excessive clearance associated with parenteral delivery. This review investigates (1) whether intravenous nanoformulations improve curcumin pharmacokinetics (PK) and (2) whether improved PK yields greater therapeutic efficacy. Standard PK parameters (measured maximum concentration [Cmax], area under the curve [AUC], distribution volume [Vd], and clearance [CL]) of intravenously administered free curcumin in mice and rats were sourced from literature and compared to curcumin formulated in nanoparticles, micelles, and liposomes. The studies that also featured analysis of pharmacodynamics (PD) in murine cancer models were used to determine whether improved PK of nanoencapsulated curcumin resulted in improved PD. The distribution and clearance of free and nanoformulated curcumin were very fast, typically accounting for >80% curcumin elimination from plasma within 60 min. Case-matched analysis demonstrated that curcumin nanoencapsulation generally improved curcumin PK in terms of measured Cmax (n = 27) and AUC (n = 33), and to a lesser extent Vd and CL. However, when the data were unpaired and clustered for comparative analysis, only 5 out of the 12 analyzed nanoformulations maintained a higher relative curcumin concentration in plasma over time compared to free curcumin. Quantitative analysis of the mean plasma concentration of free curcumin versus nanoformulated curcumin did not reveal an overall marked improvement in curcumin PK. No correlation was found between PK and PD, suggesting that augmentation of the systemic presence of curcumin does not necessarily lead to greater therapeutic efficacy.

Magnetic beads for the evaluation of drug release from biotinylated polymeric micelles in biological media.

To improve the reliability of in vitro release studies of drug delivery systems, we developed a novel in vitro method for the evaluation of drug release from polymeric micelles in complex biological media. Polymeric micelles based on poly(N-2-hydroxypropyl methacrylamide)-block-poly(N-2-benzoyloxypropyl methacrylamide) (p(HPMAm)-b-p(HPMAm-Bz)) of which 10% of the chains was functionalized with biotin at the p(HPMAm) terminus were prepared using a solvent extraction method. The size of the micelles when loaded with a hydrophobic agent, namely paclitaxel (a clinically used cytostatic drug) or curcumin (a compound with multiple pharmacological activities), was around 65 nm. The biotin decoration allowed the binding of the micelles to streptavidin-coated magnetic beads which occurred within 10 min and reached a binding efficiency of 90 ± 6%. Drug release in different media was studied after the magnetic separation of micelles bound to the streptavidin-coated beads, by determination of the released drug in the media as well as the retained drug in the micellar fraction bound to the beads. The in vitro release of paclitaxel and curcumin at 37 °C in PBS, PBS containing 2% v/v Tween 80, PBS containing 4.5% w/v bovine serum albumin, mouse plasma, and whole mouse blood was highly medium-dependent. In all media studied, paclitaxel showed superior micellar retention compared to curcumin. Importantly, the presence of serum proteins accelerated the release of both paclitaxel and curcumin. The results presented in this study show great potential for predicting drug release from nanomedicines in biological media which in turn is crucial for their further pharmaceutical development.

Experimental Conditions That Influence the Utility of 2'7'-Dichlorodihydrofluorescein Diacetate (DCFH2-DA) as a Fluorogenic Biosensor for Mitochondrial Redox Status.

Oxidative stress has been causally linked to various diseases. Electron transport chain (ETC) inhibitors such as rotenone and antimycin A are frequently used in model systems to study oxidative stress. Oxidative stress that is provoked by ETC inhibitors can be visualized using the fluorogenic probe 2',7'-dichlorodihydrofluorescein-diacetate (DCFH2-DA). Non-fluorescent DCFH2-DA crosses the plasma membrane, is deacetylated to 2',7'-dichlorodihydrofluorescein (DCFH2) by esterases, and is oxidized to its fluorescent form 2',7'-dichlorofluorescein (DCF) by intracellular ROS. DCF fluorescence can, therefore, be used as a semi-quantitative measure of general oxidative stress. However, the use of DCFH2-DA is complicated by various protocol-related factors that mediate DCFH2-to-DCF conversion independently of the degree of oxidative stress. This study therefore analyzed the influence of ancillary factors on DCF formation in the context of ETC inhibitors. It was found that ETC inhibitors trigger DCF formation in cell-free experiments when they are co-dissolved with DCFH2-DA. Moreover, the extent of DCF formation depended on the type of culture medium that was used, the pH of the assay system, the presence of fetal calf serum, and the final DCFH2-DA solvent concentration. Conclusively, experiments with DCFH2-DA should not discount the influence of protocol-related factors such as medium and mitochondrial inhibitors (and possibly other compounds) on the DCFH2-DA-DCF reaction and proper controls should always be built into the assay protocol.

CpG-binding protein CFP1 promotes ovarian cancer cell proliferation by regulating BST2 transcription.

Epigenetic alterations have been functionally linked to ovarian cancer development and occurrence. The CXXC zinc finger protein 1 (CFP1) is an epigenetic regulator involved in DNA methylation and histone modification in mammalian cells. However, its role in ovarian cancer cells is unknown. Here, we show that CFP1 protein is highly expressed in human ovarian cancer tissues. Loss of CFP1 inhibited the growth of human ovarian cancer cells, promoted apoptosis, and increased senescence. CFP1 knockdown resulted in reduced levels of SETD1 (a CFP1 partner) and histone H3 trimethylation at the fourth lysine residue (H3K4me3). RNA-sequencing revealed that deletion of CFP1 resulted in mRNA reduction of bone marrow stromal cell antigen 2 (BST2). Bioinformatics analysis and chromatin immunoprecipitation showed that CFP1 binds to the promoter of BST2 and regulates its transcription directly. Overexpression of BST2 rescued the growth inhibitory effect of CFP1 loss. Furthermore, depletion of cullin-RING ubiquitin ligases 4 (CRL4) components ROC1 or CUL4A had significantly inhibited the expression of CFP1 and BST2 similar to MLN4924 treatment that blocked cullin neddylation and inactivated CRL4s. In conclusion, CFP1 promotes ovarian cancer cell proliferation and apoptosis by regulating the transcription of BST2, and the expression of CFP1 was affected by CRL4 ubiquitin ligase complex.

Metallated phthalocyanines and their hydrophilic derivatives for multi-targeted oncological photodynamic therapy.

BACKGROUND AND AIM: A photosensitizer (PS) delivery and comprehensive tumor targeting platform was developed that is centered on the photosensitization of key pharmacological targets in solid tumors (cancer cells, tumor vascular endothelium, and cellular and non-cellular components of the tumor microenvironment) before photodynamic therapy (PDT). Interstitially targeted liposomes (ITLs) encapsulating zinc phthalocyanine (ZnPC) and aluminum phthalocyanine (AlPC) were formulated for passive targeting of the tumor microenvironment. In previous work it was established that the PEGylated ITLs were taken up by cultured cholangiocarcinoma cells. The aim of this study was to verify previous results in cancer cells and to determine whether the ITLs can also be used to photosensitize cells in the tumor microenvironment and vasculature. Following positive results, rudimentary in vitro and in vivo experiments were performed with ZnPC-ITLs and AlPC-ITLs as well as their water-soluble tetrasulfonated derivatives (ZnPCS4 and AlPCS4) to assemble a research dossier and bring this platform closer to clinical transition. METHODS: Flow cytometry and confocal microscopy were employed to determine ITL uptake and PS distribution in cholangiocarcinoma (SK-ChA-1) cells, endothelial cells (HUVECs), fibroblasts (NIH-3T3), and macrophages (RAW 264.7). Uptake of ITLs by endothelial cells was verified under flow conditions in a flow chamber. Dark toxicity and PDT efficacy were determined by cell viability assays, while the mode of cell death and cell cycle arrest were assayed by flow cytometry. In vivo systemic toxicity was assessed in zebrafish and chicken embryos, whereas skin phototoxicity was determined in BALB/c nude mice. A PDT efficacy pilot was conducted in BALB/c nude mice bearing human triple-negative breast cancer (MDA-MB-231) xenografts. RESULTS: The key findings were that (1) photodynamically active PSs (i.e., all except ZnPCS4) were able to effectively photosensitize cancer cells and non-cancerous cells; (2) following PDT, photodynamically active PSs were highly toxic-to-potent as per anti-cancer compound classification; (3) the photodynamically active PSs did not elicit notable systemic toxicity in zebrafish and chicken embryos; (4) ITL-delivered ZnPC and ZnPCS4 were associated with skin phototoxicity, while the aluminum-containing PSs did not exert detectable skin phototoxicity; and (5) ITL-delivered ZnPC and AlPC were equally effective in their tumor-killing capacity in human tumor breast cancer xenografts and superior to other non-phthalocyanine PSs when appraised on a per mole administered dose basis. CONCLUSIONS: AlPC(S4) are the safest and most effective PSs to integrate into the comprehensive tumor targeting and PS delivery platform. Pending further in vivo validation, these third-generation PSs may be used for multi-compartmental tumor photosensitization.

Strategies for Improving Photodynamic Therapy Through Pharmacological Modulation of the Immediate Early Stress Response.

Photodynamic therapy (PDT) is a minimally to noninvasive treatment modality that has emerged as a promising alternative to conventional cancer treatments. PDT induces hyperoxidative stress and disrupts cellular homeostasis in photosensitized cancer cells, resulting in cell death and ultimately removal of the tumor. However, various survival pathways can be activated in sublethally afflicted cancer cells following PDT. The acute stress response is one of the known survival pathways in PDT, which is activated by reactive oxygen species and signals via ASK-1 (directly) or via TNFR (indirectly). The acute stress response can activate various other survival pathways that may entail antioxidant, pro-inflammatory, angiogenic, and proteotoxic stress responses that culminate in the cancer cell's ability to cope with redox stress and oxidative damage. This review provides an overview of the immediate early stress response in the context of PDT, mechanisms of activation by PDT, and molecular intervention strategies aimed at inhibiting survival signaling and improving PDT outcome.

Inhibition of the HIF-1 Survival Pathway as a Strategy to Augment Photodynamic Therapy Efficacy.

Photodynamic therapy (PDT) is a non-to-minimally invasive treatment modality that utilizes photoactivatable drugs called photosensitizers to disrupt tumors with locally photoproduced reactive oxygen species (ROS). Photosensitizer activation by light results in hyperoxidative stress and subsequent tumor cell death, vascular shutdown and hypoxia, and an antitumor immune response. However, sublethally afflicted tumor cells initiate several survival mechanisms that account for decreased PDT efficacy. The hypoxia inducible factor 1 (HIF-1) pathway is one of the most effective cell survival pathways that contributes to cell recovery from PDT-induced damage. Several hundred target genes of the HIF-1 heterodimeric complex collectively mediate processes that are involved in tumor cell survival directly and indirectly (e.g., vascularization, glucose metabolism, proliferation, and metastasis). The broad spectrum of biological ramifications culminating from the activation of HIF-1 target genes reflects the importance of HIF-1 in the context of therapeutic recalcitrance. This chapter elaborates on the involvement of HIF-1 in cancer biology, the hypoxic response mechanisms, and the role of HIF-1 in PDT. An overview of inhibitors that either directly or indirectly impede HIF-1-mediated survival signaling is provided. The inhibitors may be used as pharmacological adjuvants in combination with PDT to augment therapeutic efficacy.

Super-Resolution Imaging of Intracellular Lipid Nanocarriers to Study Drug Delivery in Photodynamic Therapy.

Liposomal nanocarriers are intensively investigated as delivery vehicles for photoactivatable agents used in photodynamic therapy (PDT). The uptake, intracellular distribution, and processing of the nanocarriers are of paramount importance for the effectiveness of the therapy; visualization and analysis of these processes can, therefore, stimulate the development of improved PDT modalities. Here we describe a simple protocol, based on super-resolution imaging, that can be used for detailed quantification of concentration, distribution, and size of individual lipid nanocarriers in adherent mammalian cells.

Optimal Use of 2',7'-Dichlorofluorescein Diacetate in Cultured Hepatocytes.

Oxidative stress is a state that arises when the production of reactive transients overwhelms the cell's capacity to neutralize the oxidants and radicals. This state often coincides with the pathogenesis and perpetuation of numerous chronic diseases. On the other hand, medical interventions such as radiation therapy and photodynamic therapy generate radicals to selectively damage and kill diseased tissue. As a result, the qualification and quantification of oxidative stress are of great interest to those studying disease mechanisms as well as therapeutic interventions. 2',7'-Dichlorodihydrofluorescein-diacetate (DCFH2-DA) is one of the most widely used fluorogenic probes for the detection of reactive transients. The nonfluorescent DCFH2-DA crosses the plasma membrane and is deacetylated by cytosolic esterases to 2',7'-dichlorodihydrofluorescein (DCFH2). The nonfluorescent DCFH2 is subsequently oxidized by reactive transients to form the fluorescent 2',7'-dichlorofluorescein (DCF). The use of DCFH2-DA in hepatocyte-derived cell lines is more challenging because of membrane transport proteins that interfere with probe uptake and retention, among several other reasons. Cancer cells share some of the physiological and biochemical features with hepatocytes, so probe-related technical issues are applicable to cultured malignant cells as well. This study therefore analyzed the in vitro properties of DCFH2-DA in cultured human hepatocytes (HepG2 cells and differentiated and undifferentiated HepaRG cells) to identify methodological and technical features that could impair proper data analysis and interpretation. The main issues that were found and should therefore be accounted for in experimental design include the following: (1) both DCFH2-DA and DCF are taken up rapidly, (2) DCF is poorly retained in the cytosol and exits the cell, (3) the rate of DCFH2 oxidation is cell type-specific, (4) DCF fluorescence intensity is pH-dependent at pH < 7, and (5) the stability of DCFH2-DA in cell culture medium relies on medium composition. Based on the findings, the conditions for the use of DCFH2-DA in hepatocyte cell lines were optimized. Finally, the optimized protocol was reduced to practice and DCFH2-DA was applied to visualize and quantify oxidative stress in real time in HepG2 cells subjected to anoxia/reoxygenation as a source of reactive transients.

DCAF13 promotes breast cancer cell proliferation by ubiquitin inhibiting PERP expression.

Evolutionarily conserved DDB1-and CUL4-associated factor 13 (DCAF13) is a recently discovered substrate receptor for the cullin RING-finger ubiquitin ligase 4 (CRL4) E3 ubiquitin ligase that regulates cell cycle progression. DCAF13 is overexpressed in many cancers, although its role in breast cancer is currently elusive. In this study we demonstrate that DCAF13 is overexpressed in human breast cancer and that its overexpression closely correlates with poor prognosis, suggesting that DCAF13 may serve as a diagnostic marker and therapeutic target. We knocked down DCAF13 in breast cancer cell lines using CRISPR/Cas9 and found that DCAF13 deletion markedly reduced breast cancer cell proliferation, clone formation, and migration both in vitro and in vivo. In addition, DCAF13 deletion promoted breast cancer cell apoptosis and senescence, and induced cell cycle arrest in the G1/S phase. Genome-wide RNAseq analysis and western blotting revealed that loss of DCAF13 resulted in both mRNA and protein accumulation of p53 apoptosis effector related to PMP22 (PERP). Knockdown of PERP partially reversed the hampered cell proliferation induced by DCAF13 knockdown. Co-immunoprecipitation assays revealed that DCAF13 and DNA damage-binding protein 1 (DDB1) directly interact with PERP. Overexpression of DDB1 significantly increased PERP polyubiquitination, suggesting that CRL4DCAF13 E3 ligase targets PERP for ubiquitination and proteasomal degradation. In conclusion, DCAF13 and the downstream effector PERP occupy key roles in breast cancer proliferation and potentially serve as prognostics and therapeutic targets.

Liver Injury and Acute Liver Failure After Bariatric Surgery: An Overview of Potential Injury Mechanisms.

The obesity epidemic has caused a surge in the use of bariatric surgery. Although surgery-induced weight loss is an effective treatment of nonalcoholic fatty liver disease, it may precipitate severe hepatic complications under certain circumstances. Acute liver injury (ALI) and acute liver failure (ALF) following bariatric surgery have been reported in several case series. Although rare, ALI and ALF tend to emerge several months after bariatric surgery. If so, it can result in prolonged hospitalization, may necessitate liver transplantation, and in some cases prove fatal. However, little is known about the risk factors for developing ALI or ALF after bariatric surgery and the mechanisms of liver damage in this context are poorly defined. This review provides an account of the available data on ALI and ALF caused by bariatric surgery, with emphasis on potential injury mechanisms and the outcomes of liver transplantation for ALF after bariatric surgery.