Heterologous mRNA-protein vaccination with Tc24 induces a robust cellular immune response against Trypanosoma cruzi, characterized by an increased level of polyfunctional CD8+ T-cells.

Tc24 is a Trypanosoma cruzi-derived flagellar protein that, when formulated with a TLR-4 agonist adjuvant, induces a balanced immune response in mice, elevating IgG2a antibody titers and IFN-γ levels. Furthermore, vaccination with the recombinant Tc24 protein can reduce parasite levels and improve survival during acute infection. Although some mRNA vaccines have been proven to elicit a stronger immune response than some protein vaccines, they have not been used against T. cruzi. This work evaluates the immunogenicity of a heterologous prime/boost vaccination regimen using protein and mRNA-based Tc24 vaccines. Mice (C57BL/6) were vaccinated twice subcutaneously, three weeks apart, with either the Tc24-C4 protein + glucopyranosyl A (GLA)-squalene emulsion, Tc24 mRNA Lipid Nanoparticles, or with heterologous protein/mRNA or mRNA/protein combinations, respectively. Two weeks after the last vaccination, mice were euthanized, spleens were collected to measure antigen-specific T-cell responses, and sera were collected to evaluate IgG titers and isotypes. Heterologous presentation of the Tc24 antigen generated antigen-specific polyfunctional CD8+ T cells, a balanced Th1/Th2/Th17 cytokine profile, and a balanced humoral response with increased serum IgG, IgG1 and IgG2c antibody responses. We conclude that heterologous vaccination using Tc24 mRNA to prime and Tc24-C4 protein to boost induces a broad and robust antigen-specific immune response that was equivalent or superior to two doses of a homologous protein vaccine, the homologous mRNA vaccine and the heterologous Tc24-C4 Protein/mRNA vaccine.

Dorsal Spanning Plate for Perilunate Dislocations.

Purpose The purpose of this study was to compare radiographic outcomes in patients treated with the traditional method of open reduction, internal fixation (ORIF) and casting as compared with those treated with ORIF and dorsal spanning plate (DSP) fixation. We hypothesized that the application of a DSP to augment the repair of perilunate dislocations would maintain carpal stability while also allowing early loadbearing through the carpus. Materials and Methods This is a retrospective radiographic review of patients with a perilunate dislocation, who were treated with ORIF and casting or ORIF with a dorsal spanning plate between 2012-2018. Scapholunate (SL) and lunotriquetral (LT) intervals were measured immediately after the index surgery and after scheduled hardware removal. A total of 28 patients met inclusion criteria, including 13 cases with traditional treatment and 15 cases with dorsal spanning plate fixation. Results Comparison of the change in SL interval and LT interval between the 13 patients in the traditional treatment group and the 15 patients in the DSP group did not yield any clinically relevant variation after statistical analysis. Both groups demonstrated minimal change in the radiographic markers of carpal stability from postoperative radiographs obtained immediately after the index repair and after the removal of hardware. Conclusion DSP fixation placed at the index surgery with early loadbearing for the treatment of perilunate dislocation is not inferior to the current mainstay of treatment consisting of cast immobilization without loadbearing and does not confer any increased carpal instability in comparison to ORIF and casting.

Stress Shielding of Ligaments Using Nonabsorbable Suture Augmentation May Influence the Biology of Ligament Healing.

Nonabsorbable suture augmentation of ligament reconstruction has seen an increase in use over the past several years with the goal of protecting the newly reconstructed ligament while allowing early rehabilitation for a potential earlier return to activity and sport. By spanning the joint with a durable nonabsorbable suture, this construct shares the stress and load seen by the reconstructed ligament, thereby protecting it from forces that could result in an early failure during the early ligamentization phase of the tendon graft. However, stress shielding of the ligament via nonabsorbable suture augmentation is also a double-edged sword, as a reduction in the stress and load seen by the ligament during this healing phase may ultimately have an impact on the final strength and composition of the reconstructed ligament. Although the long-term effects of this stress shielding have yet to be studied or reported in human subjects, multiple biomechanical and animal studies have demonstrated overall changes in architecture, tensile strength, and mechanical properties of a stress-shielded autograft ligament reconstruction.

Mapping the Complexity of Suicide by Combining Participatory Modeling and Network Science.

Suicide rates are steadily increasing among youth in the USA. Although several theories and frameworks of suicide have been developed, they do not account for some of the features that define suicide as a complex problem, such as a large number of interrelationships and cycles. In this paper, we create the first c omprehensive m ap o f a dverse c hildhood experiences (ACEs) and suicide for youth, by combining a participatory approach (involving 15 subject-matter experts) and network science. This results in a map of 946 edges and 361 concepts, in which we identify ACEs to be the most important factor (per degree centrality). The map is openly shared with the community to support further network analyses (e.g., decomposition into clusters). Similarly to the high-impact Foresight Map developed in the context of obesity, the largest map on suicide and ACEs to date presented in this paper can start a discussion at the crossroad of suicide research and network science, thus bringing new means to address a complex public health challenge.

Electrical Impedance Tomography for monitoring cardiac radiofrequency ablation: a scoping review of an emerging technology.

Arrhythmias are common cardiac diseases which can be treated effectively by the cardiac radiofrequency ablation (CRFA). However, information regarding the lesion growth within the myocardium is critical to the procedure's safety and efficacy but still unavailable in the current catheterisation lab (CathLab). Over the last 20 years, many efforts have been made in order to track the lesion size during the procedure. Unfortunately, all the approaches have their own limitations preventing them from the clinical translation and hence making the lesion size monitoring during a CRFA still an open issue. Electrical Impedance Tomography (EIT) is an impedance imaging modality that might be able to image the thermal-related impedance changes from which the lesion size can be measured. With the availability of the patient's CT scans, for a detailed model, and the catheter-based electrodes for the internal electrodes, EIT accuracy and sensitivity to the ablated sites can be significantly improved and is worth being explored for this application. Though EIT is still new to CRFA with no in-vivo experiments being done according to our up-to-date searching, many related EIT studies and its extensive research in Hyperthermia and other ablations can reveal many hints for a possibility of the CRFA-EIT application. In this paper, we present a review on multiple aspects of EIT in CRFA. First, the expected CRFA-EIT signal range and frequency are discussed based on various measured impedance results obtained from lesions in the past. Second, the possible noise sources that can happen in a clinical CRFA procedure, along with their signal range and frequency compared to the CRFA-EIT signal, and, third, the available current solutions to separate such noises from the CRFA-EIT signal. Finally, we review the progress of EIT in thermal applications over the last two decades in order to identify the developments that EIT can take advantage of and the current drawbacks that need to be solved for a potential CRFA-EIT application.

Extensor Tendon Entrapment on Computed Tomography Imaging of Distal Radius Fractures.

Objective To examine the association between distal radius fractures and tendon entrapment identified on computed tomography (CT) imaging. Patients and Methods After Institutional Review Board approval, we retrospectively reviewed distal radius fractures that underwent CT imaging from an electronic database between January 2006 to February 2018 at a single level 1 hospital trauma center. We categorized all distal radial fractures according to the AO-OTA (AO Foundation/Orthopaedic Trauma Association) classification. Distal upper extremity tendons were assessed for entrapment. Fisher's exact test was used for statistical analysis with significance at p < 0.05. Results A total of 183 distal radius fractures were identified in 179 patients. A total of 16 fractures (13 males and 3 females) were associated with tendon entrapment. Mechanism of injury included falls ( n = 7), motor vehicle accidents ( n = 6), dog bites ( n = 2), and gunshot wound ( n = 1). Entrapped tendons were limited to the extensor compartment and included the extensor pollicis longus (EPL; n = 11), extensor pollicis brevis ( n = 1), extensor carpi ulnaris ( n = 1), extensor carpi radialis longus ( n = 1), and extensor digitorum communis ( n = 2). The most commonly associated AO-OTA fracture pattern with tendon entrapment was complete articular radial fractures (2R3C; 69%), eight of which were simple articular with metaphyseal multifragmentary fractures (2R3C2). Of the distal radius fractures, 81% were associated with additional ulnar fractures of varying severity and displacement. Conclusion Approximately 8.7% of distal radius fractures were retrospectively identified to have tendon entrapment compared with a previously reported incidence of 1.3%. Wrist surgeons and radiologists should have higher suspicion for tendon entrapment and carefully review preoperative CT imaging for tendon entrapment in distal radius fractures especially if there is an intra-articular, multifragmentary injury pattern. Wrist surgeons and radiologists should also have increased suspicion for EPL tendon entrapments given its high incidence in association with distal radius fractures. Level of Evidence This is a Level III, retrospective cross-sectional study.

Cardiac radiofrequency ablation tracking using electrical impedance tomography.

There is a need for accessible high speed imaging of Radiofrequency (RF) cardiac electrosurgery to improve safety and efficacy of the ablation time course, where lesion information is critical to safety and efficacy but currently lacking in real time. In this paper, Electrical Impedance Tomography (EIT) using existing cardiac EP electrodes was optimised to confirm (1) that removal of measurements with low signal sensitivity leads to improved images and (2) that multiple signal thresholds are needed to track the lesion accurately over time. A novel ventricle-shaped gel phantom with realistic fluid flow to mimic blood flow, lung ventilation and myocardium conductivity was developed to study the capability and motivate transition to in-vivo measurements. When using 8 external (ECG) electrodes, 4 internal coronary sinus electrodes and 4 RF catheter-based electrodes, the optimal setup for sensitivity and dynamic tracking was 77 measurements within an error of 20%. Higher thresholds were more suitable for the earlier phase of the ablation when lesions are small while lower thresholds suited later phases. Patient-specific thresholds could be optimised in pre-surgical planning where detailed anatomical images are available. While the error reported in this initial study appears large, it is a major advance over the current situation for the cardiologist where no real-time lesion visualization is accessible in a regular EP suite/cath lab.

The Relationship Between Cumulative Exogenous Corticosteroid Exposure and Volumes of Hippocampal Subfields and Surrounding Structures.

PURPOSE/BACKGROUND: Glucocorticoids are a class of hormones that include naturally occurring cortisol and corticosterone, as well as prescription drugs commonly used to manage inflammatory, autoimmune, and allergic conditions. Adverse effects, including neuropsychiatric symptoms, are common. The hippocampus appears to be especially sensitive to the effects of glucocorticoids. However, to our knowledge, no studies to date have examined hippocampal subfields in humans receiving glucocorticoids. We examined patients on chronic glucocorticoid regimens to determine relationships between dose and duration of treatment, and hippocampal subfields, and related regions volumes. METHODS/PROCEDURES: The study included adult men and women receiving at least 5 mg daily of prednisone equivalents for at least 6 months. Volumes of brain regions were measured via magnetic resonance imaging. A multivariate general linear model was used for analysis, with brain volumes as dependent variables and age, sex, and cumulative corticosteroid exposure, as predictors. FINDINGS/RESULTS: The study population consisted of 81 adult outpatients (43 male) on corticosteroids (mean dose, 7.88 mg; mean duration, 76.75 months). Cumulative glucocorticoid exposure was negatively associated with left and right hippocampal dentate gyrus/CA3 volume. In subsequent subgroup analysis, this association held true for the age group older than the median age of 46 years but not for the younger age group. IMPLICATIONS/CONCLUSIONS: This finding is consistent with previous studies showing detrimental effects of elevated glucocorticoids on the hippocampus but further suggests that the dentate gyrus and CA3 regions are particularly vulnerable to those effects, which is consistent with animal models of chronic stress but has not been previously demonstrated in humans.

Self-weighted NOSER-prior electrical impedance tomography using internal electrodes in cardiac radiofrequency ablation.

OBJECTIVE: Radiofrequency (RF) cardiac ablation is a commonly used method for treating cardiac arrhythmias in which the information of the dynamic lesion heating is critical to cardiologists but is currently lacking. Electrical impedance tomography (EIT) is a temporal modality of imaging the changes in the electrical properties within a measured object and hence might be able to track the electrical variation due to temperature changes within the myocardium. Within this paper, (1) a time-efficient algorithm with self-weighted NOSER-prior and (2) a measurement filtering process for optimizing the number of measurement were proposed for monitoring the lesion size during the cardiac RF ablation, taking advantage of internal catheter-based electrodes and the prior information of anatomical structure and the catheter location, which are usually available during the ablation course. APPROACH: A tank model with a circular myocardium of 12 mm in thickness, 16 external electrodes on the boundary and three internal catheter-based electrodes positioned inside the endocardium were made. The ablations were simulated using Pennes' bioheat transfer equation and the simulated temperature gradients were then transferred to EIT measurements. The algorithm used one reference ablation for its optimization and then was tested with numerous 90 s ablations containing three disturbances: the catheter location mapping, the wide range of varied myocardium conductivity and the blood's cooling convection, and the Gaussian noises with 10-40 µV in standard deviation. MAIN RESULTS: The results showed that, with the optimized number of 55 measurements, the algorithm still performed well when dealing with all three disturbances plus the random noises up 25 µV. Specifically, the lesion depth and width were measured within 1.6 mm and 3.2 mm in error respectively in at least 80% out of 100 simulated ablations. SIGNIFICANCE: The algorithm has successfully measured the lesion size with good accuracy and tolerances of noise and other system perturbations. More tests in vitro and in vivo are required in the future to confirm the algorithm's feasibility.

Benign Bone Tumors When They Coexist with Common Orthopaedic Conditions.

Benign and malignant bone tumors around the knee can be encountered with concomitant orthopaedic disease. As such, it is important to recognize these oncologic processes that may impact or alter the routine treatment of more common orthopaedic processes. Frequently, these rare oncologic conditions are discovered only through the evaluation of acute injuries as in the setting of fractures or sports injuries. Other times, they are encountered when evaluating chronic entities, such as arthroplasty. This article aims to provide general guidelines and management strategies for benign bone tumors when they coincide with common orthopaedic conditions around the knee.

Heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by increasing protein translation of selected transcripts in cancer cells.

The heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by coordinating the translation of selected transcripts associated with proliferation and survival. hnRNP A18 binds to and stabilizes the transcripts of pro-survival genes harboring its RNA signature motif in their 3'UTRs. hnRNP A18 binds to ATR, RPA, TRX, HIF-1α and several protein translation factor mRNAs on polysomes and increases de novo protein translation under cellular stress. Most importantly, down regulation of hnRNP A18 decreases proliferation, invasion and migration in addition to significantly reducing tumor growth in two mouse xenograft models, melanoma and breast cancer. Moreover, tissue microarrays performed on human melanoma, prostate, breast and colon cancer indicate that hnRNP A18 is over expressed in 40 to 60% of these malignant tissue as compared to normal adjacent tissue. Immunohistochemistry data indicate that hnRNP A18 is over expressed in the stroma and hypoxic areas of human tumors. These data thus indicate that hnRNP A18 can promote tumor growth in in vivo models by coordinating the translation of pro-survival transcripts to support the demands of proliferating cells and increase survival under cellular stress. hnRNP A18 therefore represents a new target to selectively inhibit protein translation in tumor cells.

Contribution of Dual Oxidase 2 (DUOX2) to Hyper-Radiosensitivity in Human Gastric Cancer Cells.

Whole-abdominal radiotherapy (WART) is a primary method for managing gastrointestinal cancers that have disseminated into intra-abdominal tissues. While effective, this approach is limited because of the increased toxicity to normal tissue associated with combined WART and full-dose chemotherapy regimens. Recent studies have demonstrated a survival advantage in a novel treatment paradigm that allows for the safe use of full-dose systemic chemotherapy in combination with low-dose fractionated radiotherapy (LDFRT). Traditionally, radiation doses greater than 120 cGy have been used in radiotherapy because lower doses were thought to be ineffective for tumor therapy. However, we now know that LDFRT can produce hyper-radiosensitivity (HRS), a phenomenon where cells undergo apoptosis at radiation doses as low as 15 cGy, in a number of proliferating cells. The objectives of our current study were to determine whether LDFRT can induce HRS in gastrointestinal cancer cells and to identify biomarkers of chemopotentiation by LDFRT. Our data indicate that three consecutive daily fractions of 15 cGy produced HRS in gastric cancer cells and potentiated a modified regimen of docetaxel, cisplatin and 5'-fluorouracil (mDCF). Colony survival assays indicated that 15 cGy was sufficient to kill 90% of the cells when LDFRT was combined with mDCF whereas a dose almost 10 times higher (135 cGy) was needed to achieve the same rate when using conventional radiotherapy alone. RT(2) PCR Profiler™ array analysis indicated that this combined regimen upregulated dual oxidase 2 (DUOX2), an enzyme functioning in the production of hydrogen peroxide, without upregulating genes involved in DNA repair. Moreover, downregulation of DUOX2 increased radioresistance at every radiation dose tested. In addition, our data indicate that reactive oxygen species (ROS) increase up to 3.5-fold in cells exposed to LDFRT and mDCF. Furthermore, inhibition of NADPH oxidase abrogated the killing efficiency of this combined regimen. Taken together these data suggest that chemopotentiation by LDFRT in gastric cancer cells may be due, at least in part, to increased ROS production (DUOX2) without upregulation of the DNA repair machinery. These data thus provide a rationale for further explorations of potential clinical applications of LDFRT, such as in WART, as a chemopotentiator for advanced and metastatic gastric cancers.

AroER tri-screen™ is a novel functional assay to estimate both estrogenic and estrogen precursor activity of chemicals or biological specimens.

The purpose of the study is to define AroER tri-screen's utility for identifying endocrine-disrupting chemicals (EDCs) that target aromatase and/or estrogen receptor (ER), and to measure the total estrogenic activity in biological specimens. ER-positive, aromatase-expressing MCF-7 breast cancer cells were stably transfected with an estrogen responsive element (ERE)-driven luciferase reporter plasmid to yield a new high-throughput screening platform-the AroER tri-screen. AroER tri-screen was capable of identifying estrogen precursors, such as cortodoxone, which function as estrogens through a two-step conversion process in aromatase-expressing tissue. Furthermore, the system proved useful for assessing EDC activity in biologically relevant samples. Estimating these activities is critical because natural estrogens and estrogenic EDCs are important factors in ER-positive breast cancer risk. As our research demonstrates, incorporating functionally active aromatase into the AroER tri-screen produces a powerful and unique tool to (1) identify new EDCs targeting aromatase and/or ER; (2) discover novel EDCs activated by aromatase; and (3) estimate overall estrogenic activities in biological samples as a potential intermediate risk factor for breast cancer.

mTOR kinase inhibitors synergize with histone deacetylase inhibitors to kill B-cell acute lymphoblastic leukemia cells.

High activity of the mechanistic target of rapamycin (mTOR) is associated with poor prognosis in pre-B-cell acute lymphoblastic leukemia (B-ALL), suggesting that inhibiting mTOR might be clinically useful. However, emerging data indicate that mTOR inhibitors are most effective when combined with other target agents. One strategy is to combine with histone deacetylase (HDAC) inhibitors, since B-ALL is often characterized by epigenetic changes that silence the expression of pro-apoptotic factors. Here we tested combinations of mTOR and pan-HDAC inhibitors on B-ALL cells, including both Philadelphia chromosome-positive (Ph+) and non-Ph cell lines. We found that mTOR kinase inhibitors (TOR-KIs) synergize with HDAC inhibitors to cause apoptosis in B-ALL cells and the effect is greater when compared to rapamycin plus HDAC inhibitors. The combination of TOR-KIs with the clinically approved HDAC inhibitor vorinostat increased apoptosis in primary pediatric B-ALL cells in vitro. Mechanistically, TOR-KI and HDAC inhibitor combinations increased expression of pro-death genes, including targets of the Forkhead Box O (FOXO) transcription factors, and increased sensitivity to apoptotic triggers at the mitochondria. These findings suggest that targeting epigenetic factors can unmask the cytotoxic potential of TOR-KIs towards B-ALL cells.

Nano-chemical infrared imaging of membrane proteins in lipid bilayers.

The spectroscopic characterization of biomolecular structures requires nanometer spatial resolution and chemical specificity. We perform full spatio-spectral imaging of dried purple membrane patches purified from Halobacterium salinarum with infrared vibrational scattering-type scanning near-field optical microscopy (s-SNOM). Using near-field spectral phase contrast based on the Amide I resonance of the protein backbone, we identify the protein distribution with 20 nm spatial resolution and few-protein sensitivity. This demonstrates the general applicability of s-SNOM vibrational nanospectroscopy, with potential extension to a wide range of biomolecular systems.

Atomic force microscopy with sub-picoNewton force stability for biological applications.

Atomic force microscopy (AFM) is widely used in the biological sciences. Despite 25 years of technical developments, two popular modes of bioAFM, imaging and single molecule force spectroscopy, remain hindered by relatively poor force precision and stability. Recently, we achieved both sub-pN force precision and stability under biologically useful conditions (in liquid at room temperature). Importantly, this sub-pN level of performance is routinely accessible using a commercial cantilever on a commercial instrument. The two critical results are that (i) force precision and stability were limited by the gold coating on the cantilevers, and (ii) smaller yet stiffer cantilevers did not lead to better force precision on time scales longer than 25 ms. These new findings complement our previous work that addressed tip-sample stability. In this review, we detail the methods needed to achieve this sub-pN force stability and demonstrate improvements in force spectroscopy and imaging when using uncoated cantilevers. With this improved cantilever performance, the widespread use of nonspecific biomolecular attachments becomes a limiting factor in high-precision studies. Thus, we conclude by briefly reviewing site-specific covalent-immobilization protocols for linking a biomolecule to the substrate and to the AFM tip.

Routine and timely sub-picoNewton force stability and precision for biological applications of atomic force microscopy.

Force drift is a significant, yet unresolved, problem in atomic force microscopy (AFM). We show that the primary source of force drift for a popular class of cantilevers is their gold coating, even though they are coated on both sides to minimize drift. Drift of the zero-force position of the cantilever was reduced from 900 nm for gold-coated cantilevers to 70 nm (N = 10; rms) for uncoated cantilevers over the first 2 h after wetting the tip; a majority of these uncoated cantilevers (60%) showed significantly less drift (12 nm, rms). Removing the gold also led to ∼10-fold reduction in reflected light, yet short-term (0.1-10 s) force precision improved. Moreover, improved force precision did not require extended settling; most of the cantilevers tested (9 out of 15) achieved sub-pN force precision (0.54 ± 0.02 pN) over a broad bandwidth (0.01-10 Hz) just 30 min after loading. Finally, this precision was maintained while stretching DNA. Hence, removing gold enables both routine and timely access to sub-pN force precision in liquid over extended periods (100 s). We expect that many current and future applications of AFM can immediately benefit from these improvements in force stability and precision.

Rapid and profound potentiation of Apo2L/TRAIL-mediated cytotoxicity and apoptosis in thoracic cancer cells by the histone deacetylase inhibitor Trichostatin A: the essential role of the mitochondria-mediated caspase activation cascade.

Apo2L/TRAIL is actively investigated as a novel targeted agent to directly induce apoptosis of susceptible cancer cells. Apo2L/TRAIL-refractory cells can be sensitized to the cytotoxic effect of this ligand by cytotoxic chemotherapeutics. The aim of this study was to evaluate the in vitro tumoricidal activity of the Apo2L/TRAIL + Trichostatin A in cultured thoracic cancer cells and to elucidate the molecular basis of the synergistic cytotoxicity of this combination. Concurrent exposure of cultured cancer cells to sublethal concentrations of Apo2L/TRAIL and Trichostatin A resulted in profound enhancement of Apo2L/TRAIL-mediated cytotoxicity in all cell lines regardless of their intrinsic susceptibility to this ligand. This combination was not toxic to primary normal cells. While Apo2L/TRAIL alone or Trichostatin A alone mediated < 20% cell death, 60 to 90% of cancer cells were apoptotic following treatment with TSA + Apo2L/TRAIL combinations. Complete translocation of Bax from the cytosol to the mitochondria compartment was mainly observed in combination-treated cells and this was correlated with robust elevation of caspase 9 proteolytic activity indicative of activation of the mitochondria apoptogenic effect. Profound TSA + Apo2L/TRAIL-mediated cytotoxicity and apoptosis were completely abrogated by either Bcl2 over-expression or by the selective caspase 9 inhibitor, highlighting the essential role of mitochondria-dependent apoptosis signaling cascade in this process. Moreover, increased caspase 8 activity observed in cells treated with the TSA + Apo2L/TRAIL combination was completely suppressed by Bcl-2 over-expression or by the selective caspase 9 inhibitor indicating that the elevated caspase 8 activity in combination-treated cells was secondary to a mitochondria-mediated amplification feedback loop of caspase activation. These finding form the basis for further development of HDAC inhibitors + Apo2L/TRAIL combination as novel targeted therapy for thoracic malignancies.

Gossypol, a phytochemical with BH3-mimetic property, sensitizes cultured thoracic cancer cells to Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand.

OBJECTIVES: Chemotherapeutic agents sensitize cancer cells to Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) via recruitment of the mitochondria-dependent activation of caspase and induction of apoptosis. This study was designed to evaluate whether gossypol, a phytochemical compound with BH3-mimetic property that functions as an inhibitor of Bcl2/BclXL, would sensitize cultured thoracic cancer cells to this death-inducing ligand. METHODS: Cancer cell lines from the lung (H460, H322), the esophagus (TE2, TE12), and the pleura (H290, H211) or primary normal cells were treated with gossypol+Apo2L/TRAIL combinations. Cell viability and apoptosis were evaluated by (4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assays, respectively. Caspase 9 and 3 specific proteolytic activity in combination-treated cells was determined by fluorometric enzymatic assay. RESULTS: Gossypol, selectively cytotoxic to cancer cells and not primary normal cells, significantly sensitized thoracic cancer cells to Apo2L/TRAIL as indicated by 1.5- to more than 10-fold reduction of Apo2L/TRAIL 50% inhibitory concentration values in cells treated with gossypol+Apo2L/TRAIL combinations. Whereas less than 20% of cancer cells exposed to either gossypol (5 micromol/L) or Apo2L/TRAIL (20 ng/mL) were dead, more than 90% of cells treated with the drug combinations were apoptotic. Combination-induced cytotoxicity and apoptosis was completely abrogated either by overexpression of Bcl2 or by the selective caspase 9 inhibitor. This combination was not toxic to normal cells. CONCLUSION: Gossypol profoundly sensitizes thoracic cancer cells to the cytotoxic effect of Apo2L/TRAIL via activation of the mitochondria-dependent death signaling pathway. This study provides evidence for the profound anticancer activity of this drug combination and should be further evaluated as a novel targeted molecular therapeutic for thoracic cancers.

Enhancement of Apo2L/TRAIL-mediated cytotoxicity in esophageal cancer cells by cisplatin.

Although expressing adequate levels of functional tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors DR4/DR5, significant proportion of cancer cells exhibit resistance to the cytotoxic effect of this ligand. Exposure of Apo2L/TRAIL-refractory cancer cells to cytotoxic chemotherapeutic agents enhances their sensitivity to Apo2L/TRAIL cytotoxicity. This study aims to elucidate the molecular mechanism responsible for the cisplatin-mediated enhancement of Apo2L/TRAIL sensitivity in cultured esophageal cancer cells. Exposure of cancer cells to sublethal concentrations of cisplatin resulted in profound potentiation of their susceptibility to Apo2L/TRAIL cytotoxicity as indicated by 2- to >20-fold reduction in Apo2L/TRAIL IC50 values. Significant activation of caspase-8, caspase-9, and caspase-3 was observed only in cells treated with cisplatin/Apo2L/TRAIL combination and not in those exposed to either agent alone. More importantly, activation of these key caspases was significantly abrogated by overexpression of Bcl2 or by the selective caspase-9 inhibitor. This observation strongly suggested that caspase-8 activation in cells treated with the cisplatin/Apo2L/TRAIL combination was secondary to the mitochondria-mediated amplification feedback loop and activation of the executioner caspase-3 was dependent on the recruitment of the intrinsic pathway characteristic of the type II cell. Profound combination-mediated cytotoxicity and induction of apoptosis was completely suppressed either by Bcl2 overexpression or by inhibition of caspase-9 activity, which conclusively pointed to the essential role of the mitochondria-dependent death signaling cascade in this process. Cisplatin sensitizes esophageal cancer cells to Apo2L/TRAIL cytotoxicity by potentiation of the mitochondria-dependent death signaling pathway that leads to amplification of caspase activation, particularly caspase-8, by the feedback loop to efficiently induce apoptosis.