Flower isoforms promote competitive growth in cancer.

In humans, the adaptive immune system uses the exchange of information between cells to detect and eliminate foreign or damaged cells; however, the removal of unwanted cells does not always require an adaptive immune system1,2. For example, cell selection in Drosophila uses a cell selection mechanism based on 'fitness fingerprints', which allow it to delay ageing3, prevent developmental malformations3,4 and replace old tissues during regeneration5. At the molecular level, these fitness fingerprints consist of combinations of Flower membrane proteins3,4,6. Proteins that indicate reduced fitness are called Flower-Lose, because they are expressed in cells marked to be eliminated6. However, the presence of Flower-Lose isoforms at a cell's membrane does not always lead to elimination, because if neighbouring cells have similar levels of Lose proteins, the cell will not be killed4,6,7. Humans could benefit from the capability to recognize unfit cells, because accumulation of damaged but viable cells during development and ageing causes organ dysfunction and disease8-17. However, in Drosophila this mechanism is hijacked by premalignant cells to gain a competitive growth advantage18. This would be undesirable for humans because it might make tumours more aggressive19-21. It is unknown whether a similar mechanism of cell-fitness comparison is present in humans. Here we show that two human Flower isoforms (hFWE1 and hFWE3) behave as Flower-Lose proteins, whereas the other two isoforms (hFWE2 and hFWE4) behave as Flower-Win proteins. The latter give cells a competitive advantage over cells expressing Lose isoforms, but Lose-expressing cells are not eliminated if their neighbours express similar levels of Lose isoforms; these proteins therefore act as fitness fingerprints. Moreover, human cancer cells show increased Win isoform expression and proliferate in the presence of Lose-expressing stroma, which confers a competitive growth advantage on the cancer cells. Inhibition of the expression of Flower proteins reduces tumour growth and metastasis, and induces sensitivity to chemotherapy. Our results show that ancient mechanisms of cell recognition and selection are active in humans and affect oncogenic growth.

Hand-held electron spin resonance scanner for subcutaneous oximetry using OxyChip.

PURPOSE: Electron spin resonance (ESR) is used to measure oxygen partial pressure (pO2) in biological media with many clinical applications. Traditional clinical ESR involves large magnets that encompass the subject of measurement. However, certain applications might benefit from a scanner operating within local static magnetic fields. Our group recently developed such a compact scanner for transcutaneous (surface) pO2 measurements of skin tissue. Here we extend this capability to subsurface (subcutaneous) pO2 measurements and verify it using an artificial tissue emulating (ATE) phantom. METHODS: We introduce a new scanner, tailored for subcutaneous measurements up to 2 mm beneath the skin's surface. This scanner captures pulsed ESR signals from embedded approximate 1-mm oxygen-sensing solid paramagnetic implant, OxyChip. The scanner features a static magnetic field source, producing a uniform region outside its surface, and a compact microwave resonator, for exciting and receiving ESR signals. RESULTS: ESR readings derived from an OxyChip, positioned approximately 1.5 mm from the scanner's surface, embedded in ATE phantom, exhibited a linear relation of 1/T2 versus pO2 for pO2 levels at 0, 7.6, 30, and 160 mmHg, with relative reading accuracy of about 10%. CONCLUSION: The compact ESR scanner can report pO2 data in ATE phantom from an external position relative to the scanner. Implementing this scanner in preclinical and clinical applications for subcutaneous pO2 measurements is a feasible next phase for this development. This innovative design also has the potential to operate in conjunction with artificial skin graft for wound healing, combining therapeutic and pO2 diagnostic features.

OxyChip embedded with radio-opaque gold nanoparticles for anatomic registration and oximetry in tissues.

PURPOSE: Electron paramagnetic resonance oximetry using the OxyChip as an implantable oxygen sensor can directly and repeatedly measure tissue oxygen levels. A phase I, first-in-human clinical study has established the safety and feasibility of using OxyChip for reliable and repeated measurements of oxygen levels in a variety of tumors and treatment regimens. A limitation in these studies is the inability to easily locate and identify the implanted probes in the tissue, particularly in the long term, thus limiting spatial/anatomical registration of the implant for proper interpretation of the oxygen data. In this study, we have developed and evaluated an enhanced oxygen-sensing probe embedded with gold nanoparticles (GNP), called the OxyChip-GNP, to enable visualization of the sensor using routine clinical imaging modalities. METHODS: In vitro characterization, imaging, and histopathology studies were carried out using tissue phantoms, excised tissues, and in vivo animal models (mice and rats). RESULTS: The results demonstrated a substantial enhancement of ultrasound and CT contrast using the OxyChip-GNP without compromising its electron paramagnetic resonance and oxygen-sensing properties or biocompatibility. CONCLUSIONS: The OxyChips embedded with gold nanoparticles (OxyChip-GNP) can be readily identified in soft tissues using standard clinical imaging modalities such as CT, cone beam-CT, or ultrasound imaging while maintaining its capability to make repeated in vivo measurements of tissue oxygen levels over the long term. This unique capability of the OxyChip-GNP facilitates precisely localized in vivo oxygen measurements in the clinical setting.

Aberrant expression of TMEM205 signaling promotes platinum resistance in ovarian cancer: An implication for the antitumor potential of DAP compound.

INTRODUCTION: TMEM205 is a novel transmembrane protein associated with platinum resistance (PR) in epithelial ovarian carcinoma (OC), however, the specific mechanisms associated with this resistance remain to be elucidated. METHODS: TMEM205 expression was evaluated in platinum-sensitive (PS) versus platinum resistant (PR) ovarian cancer cell lines and patient serum/tissues. Exosomal efflux of platinum was evaluated with inductively coupled plasma mass spectrometry (ICP-MS) after pre-treatment with small molecule inhibitors (L-2663/L-2797) of TMEM205 prior to treatment with platinum. Cytotoxicity of combination treatment was confirmed in vitro and in an in vivo model. RESULTS: TMEM205 expression was 10-20 fold higher in PR compared to PS ovarian cancer cell lines, serum samples, and tissues. Co-localization with CD1B was confirmed by in-situ proximity ligation assay suggesting that TMEM205 may mediate PR via the exosomal pathway. Exosomal secretion was significantly increased 5-10 fold in PR cell lines after treatment with carboplatin compared to PS cell lines. Pre-treatment with L-2663 prior to carboplatin resulted in significantly increased intracellular concentration of fluorescently-labeled cisplatin and decreased exosomal efflux of platinum. Decreased cell survival and tumor growth in vitro and in vivo was observed when PR cells were treated with a combination of L-2663 with carboplatin compared to carboplatin alone. CONCLUSION: TMEM205 appears to be involved in the development of PR in ovarian cancer through the exosomal efflux of platinum agents. This study provides pre-clinical evidence that TMEM205 could serve as a possible biomarker for PR as well as a therapeutic target in combination with platinum agents.

Compact electron spin resonance skin oximeter: Properties and initial clinical results.

PURPOSE: Skin oxygen level is of significance for the diagnosis and treatment of many clinical problems, such as chronic wounds and diabetic foot ulcers. Furthermore, skin oxygen levels can be correlated to arterial oxygen partial pressure, thereby revealing potentially dangerous conditions such as hyperoxia (too much oxygen), which may occur in ventilated neonates. Traditionally, skin oxygen levels are measured using electrochemical methods and, more recently, also by fluorescence lifetime techniques. These approaches suffer from several drawbacks, rendering them suboptimal. The purpose of this work is to develop an electron spin resonance (ESR) -based method for monitoring oxygen partial pressure (pO2 ) in skin tissue. METHODS: A compact sensor for pulsed ESR is designed and constructed. Our ESR-based method makes use of a unique exogenous paramagnetic spin probe that is placed on the skin in a special partially sealed sticker, and subsequently measuring its signal with the compact pulsed ESR sensor that includes a miniature magnet and a small S-band (~2.3 GHz) microwave resonator. The inverse of the spin-spin relaxation time (1/T2 ) measured by ESR is shown to be linearly correlated with pO2 levels. RESULTS: The sensor and its matching sticker were tested both in vitro and in vivo (with human subjects). Measured skin pO2 levels reached equilibrium after ~2-3 h and were found to be comparable to those measured by continuous-wave (CW) ESR using a large electromagnet. CONCLUSIONS: A compact pulsed ESR sensor with a matching paramagnetic sticker can be used for pO2 monitoring of the skin tissue, similar to large bulky CW ESR systems.

HIF-transcribed p53 chaperones HIF-1α.

Chronic hypoxia is associated with a variety of physiological conditions such as rheumatoid arthritis, ischemia/reperfusion injury, stroke, diabetic vasculopathy, epilepsy and cancer. At the molecular level, hypoxia manifests its effects via activation of HIF-dependent transcription. On the other hand, an important transcription factor p53, which controls a myriad of biological functions, is rendered transcriptionally inactive under hypoxic conditions. p53 and HIF-1α are known to share a mysterious relationship and play an ambiguous role in the regulation of hypoxia-induced cellular changes. Here we demonstrate a novel pathway where HIF-1α transcriptionally upregulates both WT and MT p53 by binding to five response elements in p53 promoter. In hypoxic cells, this HIF-1α-induced p53 is transcriptionally inefficient but is abundantly available for protein-protein interactions. Further, both WT and MT p53 proteins bind and chaperone HIF-1α to stabilize its binding at its downstream DNA response elements. This p53-induced chaperoning of HIF-1α increases synthesis of HIF-regulated genes and thus the efficiency of hypoxia-induced molecular changes. This basic biology finding has important implications not only in the design of anti-cancer strategies but also for other physiological conditions where hypoxia results in disease manifestation.

Oxygen-Sensing Paramagnetic Probes for Clinical Oximetry.

Electron paramagnetic resonance (EPR) oximetry based on lithium naphthalocyanine paramagnetic crystals as oxygen sensors enables direct measurement of the partial pressure of oxygen (pO2) in tissues. The method uses topical or implantable forms of these oxygen-sensing crystals embedded in a biocompatible siloxane elastomer. This article presents a summary of these sensors for EPR oximetry and their applicability for tissue oxygen measurement in the clinic.

Evaluation of a Refined Implantable Resonator for Deep-Tissue EPR Oximetry in the Clinic.

OBJECTIVES: (1) Summarize revisions made to the implantable resonator (IR) design and results of testing to characterize biocompatibility;(2) Demonstrate safety of implantation and feasibility of deep tissue oxygenation measurement using electron paramagnetic resonance (EPR) oximetry. STUDY DESIGN: In vitro testing of the revised IR and in vivo implantation in rabbit brain and leg tissues. METHODS: Revised IRs were fabricated with 1-4 OxyChips with a thin wire encapsulated with two biocompatible coatings. Biocompatibility and chemical characterization tests were performed. Rabbits were implanted with either an IR with 2 oxygen sensors or a biocompatible-control sample in both the brain and hind leg. The rabbits were implanted with IRs using a catheter-based, minimally invasive surgical procedure. EPR oximetry was performed for rabbits with IRs. Cohorts of rabbits were euthanized and tissues were obtained at 1 week, 3 months, and 9 months after implantation and examined for tissue reaction. RESULTS: Biocompatibility and toxicity testing of the revised IRs demonstrated no abnormal reactions. EPR oximetry from brain and leg tissues were successfully executed. Blood work and histopathological evaluations showed no significant difference between the IR and control groups. CONCLUSIONS: IRs were functional for up to 9 months after implantation and provided deep tissue oxygen measurements using EPR oximetry. Tissues surrounding the IRs showed no more tissue reaction than tissues surrounding the control samples. This pre-clinical study demonstrates that the IRs can be safely implanted in brain and leg tissues and that repeated, non-invasive, deep-tissue oxygen measurements can be obtained using in vivo EPR oximetry.

The curcumin analog HO-3867 selectively kills cancer cells by converting mutant p53 protein to transcriptionally active wildtype p53.

p53 is an important tumor-suppressor protein that is mutated in more than 50% of cancers. Strategies for restoring normal p53 function are complicated by the oncogenic properties of mutant p53 and have not met with clinical success. To counteract mutant p53 activity, a variety of drugs with the potential to reconvert mutant p53 to an active wildtype form have been developed. However, these drugs are associated with various negative effects such as cellular toxicity, nonspecific binding to other proteins, and inability to induce a wildtype p53 response in cancer tissue. Here, we report on the effects of a curcumin analog, HO-3867, on p53 activity in cancer cells from different origins. We found that HO-3867 covalently binds to mutant p53, initiates a wildtype p53-like anticancer genetic response, is exclusively cytotoxic toward cancer cells, and exhibits high anticancer efficacy in tumor models. In conclusion, HO-3867 is a p53 mutant-reactivating drug with high clinical anticancer potential.

Transcutaneous oxygen measurement in humans using a paramagnetic skin adhesive film.

PURPOSE: Transcutaneous oxygen tension (TcpO2 ) provides information about blood perfusion in the tissue immediately below the skin. These data are valuable in assessing wound healing problems, diagnosing peripheral vascular/arterial insufficiency, and predicting disease progression or the response to therapy. Currently, TcpO2 is primarily measured using electrochemical skin sensors, which consume oxygen and are prone to calibration errors. The goal of the present study was to develop a reliable method for TcpO2 measurement in human subjects. METHODS: We have developed a novel TcpO2 oximetry method based on electron paramagnetic resonance (EPR) principles with an oxygen-sensing skin adhesive film, named the superficial perfusion oxygen tension (SPOT) chip. The SPOT chip is a 3-mm diameter, 60-µm thick circular film composed of a stable paramagnetic oxygen sensor. The chip is covered with an oxygen-barrier material on one side and secured on the skin by a medical adhesive transfer tape to ensure that only the oxygen that diffuses through the skin surface is measured. The method quantifies TcpO2 through the linewidth of the EPR spectrum. RESULTS: Repeated measurements using a cohort of 10 healthy human subjects showed that the TcpO2 measurements were robust, reliable, and reproducible. The TcpO2 values ranged from 7.8 ± 0.8 to 22.0 ± 1.0 mmHg in the volar forearm skin (N = 29) and 8.1 ± 0.3 to 23.4 ± 1.3 mmHg in the foot (N = 86). CONCLUSIONS: The results demonstrated that the SPOT chip can measure TcpO2 reliably and repeatedly under ambient conditions. The SPOT chip method could potentially be used to monitor TcpO2 in the clinic.

Estimation of pO2 histogram from a composite EPR Spectrum of multiple random implants.

Electron paramagnetic resonance (EPR) spectroscopy using oxygen-sensing implants can provide reliable and repeated measurements of the partial pressure of oxygen (pO2) over a period of months or longer; however, it does not provide accurate information about the distribution of tissue oxygenation. While EPR imaging has the capability to provide spatially resolved oxygen data, it is time-consuming and not optimized for discrete number of implants. Previous reports suggest multi-site algorithms, which would require either the implants to be aligned in a certain way so as to deconvolve them using a linear magnetic field gradient or sparse imaging of the implants from a small number of 3D projections. In this paper, we present a simpler and much faster method to estimate the pO2 histogram from a composite, single-scan EPR spectrum measured without applying field gradients to separate the EPR signals from multiple randomly placed oxygen-sensing implants. The method is optimized for a discrete number of implants, validated using simulations, experimental phantoms and in animal models. The results established the composite spectral fitting algorithm as a reliable and robust tool for multi-site oximetry.

Implantable microchip containing oxygen-sensing paramagnetic crystals for long-term, repeated, and multisite in vivo oximetry.

EPR oximetry is established as a viable method for measuring the tissue oxygen level (partial pressure of oxygen, pO2) in animal models; however, it has not yet been established for measurements in humans. EPR oximetry requires an oxygen-sensing paramagnetic probe (molecular or particulate) to be placed at the site/organ of measurement, which may pose logistical and safety concerns, including invasiveness of the probe-placement procedure as well as lack of temporal stability and sensitivity for long-term (repeated) measurements, and possible toxicity in the short- and long-term. In the past, we have developed an implantable oxygen-sensing probe, called OxyChip, which we have successfully established for oximetry in pre-clinical animal models (Hou et al. Biomed. Microdevices 20, 29, 2018). Currently, OxyChip is being evaluated in a limited clinical trial in cancer patients. A major limitation of OxyChip is that it is a large (1.4 mm3) implant and hence not suitable for measuring oxygen heterogeneity that may be present in solid tumors, chronic wounds, etc. In this report, we describe the development of a substantially smaller version of OxyChip (0.07 mm3 or 70 cubic micron), called mChip, that can be placed in the tissue of interest using a 23G syringe-needle with minimal invasiveness. Using in vitro and in vivo models, we have shown that the microchip provides adequate EPR sensitivity, stability, and biocompatibility and thus enables robust, repeated, and simultaneous measurement from multiple implants providing mean and median pO2 values in the implanted region. The mChips will be particularly useful for those applications that require repeated measurements of mean/median pO2 in superficial tissues and malignancies.

Electron-Spin-Resonance Dipstick.

Electron spin resonance (ESR) is a powerful analytical technique used for the detection, quantification, and characterization of paramagnetic species ranging from stable organic free radicals and defects in crystals to gaseous oxygen. Traditionally, ESR requires the use of complex instrumentation, including a large magnet and a microwave resonator in which the sample is placed. Here, we present an alternative to the existing approach by inverting the typical measurement topology, namely placing the ESR magnet and resonator inside the sample rather than the other way around. This new development relies on a novel self-contained ESR sensor with a diameter of just 2 mm and length of 3.6 mm, which includes both a small permanent magnet assembly and a tiny (∼1 mm in size) resonator for spin excitation and detection at a frequency of ∼2.6 GHz. The spin sensitivity of the sensor has been measured to be ∼1011 spins/√Hz, and its concentration sensitivity is ∼0.1 mM, using reference samples with a measured volume of just ∼10 nL. Our new approach can be applied for monitoring the partial pressure of oxygen in vitro and in vivo through its paramagnetic interaction with another stable radical, as well as for simple online quantitative inspection of free radicals generated in reaction vessels and electrochemical cells via chemical processes.

Pre-clinical evaluation of OxyChip for long-term EPR oximetry.

Tissue oxygenation is a critical parameter in various pathophysiological situations including cardiovascular disease and cancer. Hypoxia can significantly influence the prognosis of solid malignancies and the efficacy of their treatment by radiation or chemotherapy. Electron paramagnetic resonance (EPR) oximetry is a reliable method for repeatedly assessing and monitoring oxygen levels in tissues. Lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) has been developed as a probe for biological EPR oximetry, especially for clinical use. However, clinical applicability of LiNc-BuO crystals is hampered by potential limitations associated with biocompatibility, biodegradation, or migration of individual bare crystals in tissue. To overcome these limitations, we have embedded LiNc-BuO crystals in polydimethylsiloxane (PDMS), an oxygen-permeable biocompatible polymer and developed an implantable/retrievable form of chip, called OxyChip. The chip was optimized for maximum spin density (40% w/w of LiNc-BuO in PDMS) and fabricated in a form suitable for implantation using an 18-G syringe needle. In vitro evaluation of the OxyChip showed that it is robust and highly oxygen sensitive. The dependence of its EPR linewidth to oxygen was linear and highly reproducible. In vivo efficacy of the OxyChip was evaluated by implanting it in rat femoris muscle and following its response to tissue oxygenation for up to 12 months. The results revealed preservation of the integrity (size and shape) and calibration (oxygen sensitivity) of the OxyChip throughout the implantation period. Further, no inflammatory or adverse reaction around the implantation area was observed thereby establishing its biocompatibility and safety. Overall, the results demonstrated that the newly-fabricated high-sensitive OxyChip is capable of providing long-term measurements of oxygen concentration in a reliable and repeated manner under clinical conditions.

Requisite Role of Kv1.5 Channels in Coronary Metabolic Dilation.

RATIONALE: In the working heart, coronary blood flow is linked to the production of metabolites, which modulate tone of smooth muscle in a redox-dependent manner. Voltage-gated potassium channels (Kv), which play a role in controlling membrane potential in vascular smooth muscle, have certain members that are redox-sensitive. OBJECTIVE: To determine the role of redox-sensitive Kv1.5 channels in coronary metabolic flow regulation. METHODS AND RESULTS: In mice (wild-type [WT], Kv1.5 null [Kv1.5(-/-)], and Kv1.5(-/-) and WT with inducible, smooth muscle-specific expression of Kv1.5 channels), we measured mean arterial pressure, myocardial blood flow, myocardial tissue oxygen tension, and ejection fraction before and after inducing cardiac stress with norepinephrine. Cardiac work was estimated as the product of mean arterial pressure and heart rate. Isolated arteries were studied to establish whether genetic alterations modified vascular reactivity. Despite higher levels of cardiac work in the Kv1.5(-/-) mice (versus WT mice at baseline and all doses of norepinephrine), myocardial blood flow was lower in Kv1.5(-/-) mice than in WT mice. At high levels of cardiac work, tissue oxygen tension dropped significantly along with ejection fraction. Expression of Kv1.5 channels in smooth muscle in the null background rescued this phenotype of impaired metabolic dilation. In isolated vessels from Kv1.5(-/-) mice, relaxation to H2O2 was impaired, but responses to adenosine and acetylcholine were normal compared with those from WT mice. CONCLUSIONS: Kv1.5 channels in vascular smooth muscle play a critical role in coupling myocardial blood flow to cardiac metabolism. Absence of these channels disassociates metabolism from flow, resulting in cardiac pump dysfunction and tissue hypoxia.

Direct and Repeated Clinical Measurements of pO2 for Enhancing Cancer Therapy and Other Applications.

The first systematic multi-center study of the clinical use of EPR oximetry has begun, with funding as a PPG from the NCI. Using particulate oxygen sensitive EPR, materials in three complementary forms (India Ink, "OxyChips", and implantable resonators) the clinical value of the technique will be evaluated. The aims include using repeated measurement of tumor pO2 to monitor the effects of treatments on tumor pO2, to use the measurements to select suitable subjects for the type of treatment including the use of hyperoxic techniques, and to provide data that will enable existing clinical techniques which provide data relevant to tumor pO2 but which cannot directly measure it to be enhanced by determining circumstances where they can give dependable information about tumor pO2.

Oxygen regulates molecular mechanisms of cancer progression and metastasis.

Oxygen is the basic molecule which supports life and it truly is "god's gift to life." Despite its immense importance, research on "oxygen biology" has never received the light of the day and has been limited to physiological and biochemical studies. It seems that in modern day biology, oxygen research is summarized in one word "hypoxia." Scientists have focused on hypoxia-induced transcriptomics and molecular-cellular alterations exclusively in disease models. Interestingly, the potential of oxygen to control the basic principles of biology like homeostatic maintenance, transcription, replication, and protein folding among many others, at the molecular level, has been completely ignored. Here, we present a perspective on the crucial role played by oxygen in regulation of basic biological phenomena. Our conclusion highlights the importance of establishing novel research areas like oxygen biology, as there is great potential in this field for basic science discoveries and clinical benefits to the society.

ErbB2 overexpression upregulates antioxidant enzymes, reduces basal levels of reactive oxygen species, and protects against doxorubicin cardiotoxicity.

Levels of the HER2/ErbB2 protein in the heart are upregulated in some women during breast cancer therapy, and these women are at high risk for developing heart dysfunction after sequential treatment with anti-ErbB2/trastuzumab or doxorubicin. Doxorubicin is known to increase oxidative stress in the heart, and thus we considered the possibility that ErbB2 protein influences the status of cardiac antioxidant defenses in cardiomyocytes. In this study, we measured reactive oxygen species (ROS) in cardiac mitochondria and whole hearts from mice with cardiac-specific overexpression of ErbB2 (ErbB2(tg)) and found that, compared with control mice, high levels of ErbB2 in myocardium result in lower levels of ROS in mitochondria (P = 0.0075) and whole hearts (P = 0.0381). Neonatal cardiomyocytes isolated from ErbB2(tg) hearts have lower ROS levels and less cellular death (P < 0.0001) following doxorubicin treatment. Analyzing antioxidant enzyme levels and activities, we found that ErbB2(tg) hearts have increased levels of glutathione peroxidase 1 (GPx1) protein (P < 0.0001) and GPx activity (P = 0.0031) in addition to increased levels of two known GPx activators, c-Abl (P = 0.0284) and Arg (P < 0.0001). Interestingly, although mitochondrial ROS emission is reduced in the ErbB2(tg) hearts, oxygen consumption rates and complex I activity are similar to control littermates. Compared with these in vivo studies, H9c2 cells transfected with ErbB2 showed less cellular toxicity and produced less ROS (P < 0.0001) after doxorubicin treatment but upregulated GR activity (P = 0.0237) instead of GPx. Our study shows that ErbB2-dependent signaling contributes to antioxidant defenses and suggests a novel mechanism by which anticancer therapies involving ErbB2 antagonists can harm myocardial structure and function.

Aberrantly activated pSTAT3-Ser727 in human endometrial cancer is suppressed by HO-3867, a novel STAT3 inhibitor.

OBJECTIVE: Constitutive activation of STAT3 is a hallmark of various human cancers, however an increased pSTAT3 expression in high grade human endometrial cancer has not been reported. In the present study, we examine the expression of STAT family of proteins in endometrial cancer cell lines and the efficacy of HO-3867, a novel STAT3 inhibitor designed in our lab. METHODS: Expression of STAT family proteins was evaluated via Western blot. The cell viability, post-treatment with HO-3867, was assessed using MTT, cell-cycle profile and Annexin assay. In vivo efficacy of HO-3867 was evaluated using xenograft mice. RESULTS: Expression of activated STATs was inconsistent among the cell lines and 18 human endometrial cancer specimens tested. While pSTAT3 Tyr705 was not expressed in any of the cell lines, pSTAT3 Ser727 was highly expressed in endometrial cancer cell lines and tumor specimens. HO-3867 decreased the expression of pSTAT3 Ser727 while total STAT3 remained constant; cell viability decreased by 50-80% and induced G2/M arrest in 55% of Ishikawa cells at the G2/M cell cycle checkpoint. There was an increase in p53, a decrease in Bcl2 and Bcl-xL, and cleavage of caspase-3, caspase-7 and PARP. HO-3867 mediated a dosage-dependent inhibition of the growth of xenografted endometrial tumors. CONCLUSIONS: HO-3867 treatment decreases the high levels of pSTAT3 Ser727 in endometrial cancer cells by inducing cell cycle arrest and apoptosis. This suggests a specific role of serine-phosphorylated STAT3, independent of tyrosine phosphorylation in the oncogenesis of endometrial cancer. HO-3867 could potentially serve as an adjunctive targeted therapy.