Majority Of Cancer Cells Research Articles

Hydralazine loaded nanodroplets combined with ultrasound-targeted microbubble destruction to induce pyroptosis for tumor treatment

Pyroptosis, a novel type of programmed cell death (PCD), which provides a feasible therapeutic option for the treatment of tumors. However, due to the hypermethylation of the promoter, the critical protein Gasdermin E (GSDME) is lacking in the majority of cancer cells, which cannot start the pyroptosis process and leads to dissatisfactory therapeutic effects. Additionally, the quick clearance, systemic side effects, and low concentration at the tumor site of conventional pyroptosis reagents restrict their use in clinical cancer therapy. Here, we described a combination therapy that induces tumor cell pyroptosis via the use of ultrasound-targeted microbubble destruction (UTMD) in combination with DNA demethylation. The combined application of UTMD and hydralazine-loaded nanodroplets (HYD-NDs) can lead to the rapid release of HYD (a demethylation drug), which can cause the up-regulation of GSDME expression, and produce reactive oxygen species (ROS) by UTMD to cleave up-regulated GSDME, thereby inducing pyroptosis. HYD-NDs combined with ultrasound (US) group had the strongest tumor inhibition effect, and the tumor inhibition rate was 87.15% (HYD-NDs group: 51.41 ± 3.61%, NDs + US group: 32.73%±7.72%), indicating that the strategy had a more significant synergistic anti-tumor effect. In addition, as a new drug delivery carrier, HYD-NDs have great biosafety, tumor targeting, and ultrasound imaging performance. According to the results, the combined therapy reasonably regulated the process of tumor cell pyroptosis, which offered a new strategy for optimizing the therapy of GSDME-silenced solid tumors.

Aglycemia induces apoptosis under hypoxic conditions in A549 cells.

Many of the cancer cells produce energy with accelerated glycolysis and perform lactic acid production even under normoxic conditions called the "Warburg effect". Metabolism can directly or indirectly regulate the apoptotic mechanism so that cancer cells take advantage of reprogrammed metabolism to avoid apoptosis. The aim of this study is to examine the mechanism of apoptosis by incubating human lung carcinoma cells (A549) under different metabolic conditions in hypoxia or normoxia environments. A549 cells were incubated in the normoxic or hypoxic condition that contained 5 mM glucose (Glc 5), 25 mM glucose (Glc 25), or 10 mM galactose (OXPHOS/aglycemic), and the mechanism of apoptosis was investigated. In the hypoxia condition, the rate of early apoptosis in aglycemic OXPHOS cells was increased (15.5% ±7.1). In addition, the activity of caspase-3 (6.1% ± 0.9), caspase-9 (30.4% ± 0.9), and cytochrome c expression level increased; however, the mitochondrial membrane potential (51.9% ± 0.4) was found to be decreased. Changing the amount of oxygen in glycolytic cells had no effect on apoptosis. However, it has been determined that apoptosis is stimulated under hypoxia conditions in aglycemic cells in which galactose is used instead of glucose. Considering that the majority of cancer cells are hypoxic, these data are important in determining targets in therapeutic intervention.

An exploratory study of cell stiffness as a mechanical label-free biomarker across multiple musculoskeletal sarcoma cells

BackgroundCancer cells are characterized by changes in cell cytoskeletal architecture and stiffness. Despite advances in understanding the molecular mechanisms of musculoskeletal cancers, the corresponding cellular mechanical properties remain largely unexplored. The aim of this study was to investigate the changes in cellular stiffness and the associated cytoskeleton configuration alterations in various musculoskeletal cancer cells.MethodsCell lines from five main sarcoma types of the musculoskeletal system (chondrosarcoma, osteosarcoma, Ewing sarcoma, fibrosarcoma and rhabdomyosarcoma) as well as their healthy cell counterparts (chondrocytes, osteoblasts, mesenchymal stem cells, fibroblasts, skeletal muscle cells) were subjected to cell stiffness measurements via atomic force microscopy (AFM). Biochemical and structural changes of the cytoskeleton (F-actin, β-tubulin and actin-related protein 2/3) were assessed by means of fluorescence labelling, ELISA and qPCR.ResultsWhile AFM stiffness measurements showed that the majority of cancer cells (osteosarcoma, Ewing sarcoma, fibrosarcoma and rhabdomyosarcoma) were significantly less stiff than their corresponding non-malignant counterparts (p < 0.001), the chondrosarcoma cells were significant stiffer than the chondrocytes (p < 0.001). Microscopically, the distribution of F-actin differed between malignant entities and healthy counterparts: the organisation in well aligned stress fibers was disrupted in cancer cell lines and the proteins was mainly concentrated at the periphery of the cell, whereas β-tubulin had a predominantly perinuclear localization. While the F-actin content was lower in cancer cells, particularly Ewing sarcoma (p = 0.018) and Fibrosarcoma (p = 0.023), this effect was even more pronounced in the case of β-tubulin for all cancer-healthy cell duos. Interestingly, chondrosarcoma cells were characterized by a significant upregulation of β-tubulin gene expression (p = 0.005) and protein amount (p = 0.032).ConclusionModifications in cellular stiffness, along with structural and compositional cytoskeleton rearrangement, constitute typical features of sarcomas cells, when compared to their healthy counterpart. Notably, whereas a decrease in stiffness is typically a feature of malignant entities, chondrosarcoma cells were stiffer than chondrocytes, with chondrosarcoma cells exhibiting a significantly upregulated β-tubulin expression. Each Sarcoma entity may have his own cellular-stiffness and cytoskeleton organisation/composition fingerprint, which in turn may be exploited for diagnostic or therapeutic purposes.

Abstract CT095: Update: a phase 1/2, open-label study to assess safety, tolerability, biodistribution, radiation dosimetry and PET imaging characteristics of [18F]FPyGal in comparison to in-vitro diagnostic for the assessment of senescence in oncological patients (NCT04536454)

Abstract It is well established that senescence of cancer cells can be induced by treatment with classical cytotoxic therapies but also by molecularly targeted therapies or immunotherapies, a phenomenon referred to as therapy induced senescence (TIS). Occurrence of intratumoral senescence harbors broad therapeutic implications and may limit the prognosis of cancer patients, as senescent cells, via their senescence associated secretory phenotype (SASP), can suppress anti-tumor immune responses and may increase the metastatic potential of non-senescent cancer cells. Preclinical data suggest that an eradication of senescent cells (senolytic therapies) or an application of SASP modulating therapies might improve the outcome of systemic cancer therapies, however, as a prerequisite to guide such approaches, robust and non-invasive modalities to visualize and quantify intratumoral senescence are needed. We recently reported on the safety evaluation of the novel Positron-Emission-Tomography (PET) tracer [18F]FPyGal, a radiolabeled substrate of senescence-associated ꞵ-galactosidase in healthy volunteers (AACR 2022, #7958). In summary, our data showed that the use of [18F]FPyGal is safe and well-tolerated in the described human study population. In the second stage of our trial, we are now investigating the concordance of histopathological senescence markers with [18F]FPyGal senescence signals in i) rectum cancer-, ii) non-small cell lung cancer- and iii) adenocarcinoma of the esophagogastric junction (AEG) patients, who are undergoing neoadjuvant therapy. Here, we report on patients from the rectum cancer cohort of the trial. [18F]FPyGal PET imaging was done before and after administration of neoadjuvant radiochemotherapy (RCTx). [18F]FPyGal PET signal was correlated with immunohistochemical senescence markers (p16, p21, p53) and quantification of SA-ß-gal (Senescence-associated beta-galactosidase) positive cells in biopsy materials (pre-therapy) and surgical resection materials (post-therapy). Expectedly, senescent cells were only detected at very low frequency in therapy naïve tumors and the lack of histological detection of senescence correlated with negativity in [18F]FPyGal imaging. In stark contrast, homogenous senescence, quantified with the indicated senescence markers, was detected in the majority of cancer cells after RCTx. Interestingly, in one rectum carcinoma, which underwent a full remission after RCTx, senescence of stroma cells in the area of the former tumor bed was detected by PET imaging and histologically. Collectively, our data indicate the utility of [18F]FPyGal PET imaging to non-invasively detect senescence. Future trials using [18F]FPyGal to guide oncological therapy, e.g. to apply senolytic drugs in a personalized manner, are warranted. Citation Format: Christian LaFougere, Brigitte Gueckel, Helmut Dittmann, Nils Trautwein, Martina Hinterleitner, Jonathan Cotton, Gerald Reischl, Gabriele Kienzle, Stephan Singer, Cihan Gani, Ulrich Lauer, Bernd Pichler, Lars Zender. Update: a phase 1/2, open-label study to assess safety, tolerability, biodistribution, radiation dosimetry and PET imaging characteristics of [18F]FPyGal in comparison to in-vitro diagnostic for the assessment of senescence in oncological patients (NCT04536454) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT095.

Cerium Oxide/Graphene Oxide Hybrid: Synthesis, Characterization, and Evaluation of Anticancer Activity in a Breast Cancer Cell Line (MCF-7)

In the present study, we used a simple ultrasonic approach to develop a Cerium oxide/Graphene oxide hybrid (CeO2/GO hybrid) nanocomposite system. Particle size analysis, Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), and X-ray Diffraction (XRD) have been used to analyze the physio-chemical characteristics of the developed nanocomposite. The synthesized hybrid system has also been examined to assess its anticancer capability against MCF-7 cell lines and normal cell lines at different sample concentrations, pH values, and incubation intervals using an antiproliferative assay test. The test results demonstrate that as sample concentration rises, the apoptotic behavior of the CeO2/GO hybrid in the MCF-7 cell line also rises. The IC50 was 62.5 µg/mL after 72 h of incubation. Cytotoxicity of cisplatin bound CeO2/GO hybrid was also tested in MCF-7 cell lines. To identify apoptosis-associated alterations of cell membranes during the process of apoptosis, a dual acridine orange/ethidium bromide (AO/EB) fluorescence staining was carried out at three specified doses (i.e., 1000 µg/mL, 250 µg/mL, and 62.5 µg/mL of CeO2/GO hybrid). The color variations from both live (green) and dead (red) cells were examined using fluorescence microscopy under in vitro conditions. The quantitative analysis was performed using flow cytometry to identify the cell cycle at which the maximum number of MCF-7 cells had been destroyed as a result of interaction with the developed CeO2/GO hybrid (FACS study). According to the results of the FACS investigation, the majority of cancer cells were inhibited at the R3 (G2/M) phase. Therefore, the CeO2/GO hybrid has successfully showed enhanced anticancer efficacy against the MCF-7 cell line at the IC50 concentration. According to the current study, the CeO2/GO platform can be used as a therapeutic platform for breast cancer. The synergetic effects of the developed CeO2/GO hybrid with the MCF-7 cell line are presented.

"Management of hospitalised adults with coronavirus disease 2019 (COVID-19): a European Respiratory Society living guideline." James D. Chalmers, Megan L. Crichton, Pieter C. Goeminne, et al. Eur Respir J 2021; 57: 2100048.

<h3>Background</h3> Cancers affect a large number of people worldwide and sometimes have mortality rates of over 90% (pancreatic ductal adenocarcinoma (PDAC), high grade glioblastomas). Currently, immunotherapeutic strategies fail in number of cancer cases due to a lack of understanding of the molecular mechanisms operated by these cancers to evade immune surveillance. Recent evidence demonstrated that the protein galectin-9 is highly expressed in majority of cancer cells. Importantly, this protein is a crucial part of the immune evasion machinery operated by human malignant tumours. Understanding the biochemistry of this immune evasion machinery is required for designing highly efficient and affordable personalised targeted immunotherapy of human cancers. <h3>Materials and Methods</h3> We used, glioblastoma, PDAC, breast and renal cancer cell lines as cancer cell models. Jurkat T lymphocytes, TALL-104 cytotoxic T cells and primary human and mouse CD3-positive T lymphocytes were employed for immune evasion studies. C57 BL16 mice were used for <i>in vivo</i> studies. Western blot analysis, on-cell and in-cell Western, ELISA, qRT-PCR, chromatin immunoprecipitation (ChIP) assays as well as a variety of biochemical tests were used to conduct the studies. <h3>Results</h3> We found that T cells trigger galectin-9 expression and secretion (in most cases requires Tim-3 protein (T cell immunoglobulin and mucine domain containing protein 3)) in a variety of human cancer cells including but not limited to glioblastoma, PDAC, breast and renal cancers. Galectin-9 triggers exhaustion and often death of cytotoxic T cells. Other immune checkpoint proteins including VISTA (V-domain Ig-containing suppressor of T cell activation) and PD-L1 (ligand of programmed cell death protein 1) promote apoptotic death of cytotoxic T cells induced by galectin-9. Activity of T helpers, required to induce cytotoxic T lymphocytes is also suppressed by galectin-9 in cooperation with VISTA and PD-L1. We discovered that expression of all these immune checkpoint proteins is differentially controlled by the autocrine transforming growth factor beta type 1 (TGF-β)-Smad3 pathway as well as several types of interferons. <h3>Conclusions</h3> The immune evasion machinery triggered by TGF-β-Smad3 and interferon pathways and operated by galectin-9, Tim-3, VISTA and PD-L1 should be considered as a potential target for personalised immunotherapy of cancer. Importantly, blood and tumour sample tests could help mapping the exact immune evasion networks employed by each tumour and the pathways responsible to produce respective immune checkpoint proteins. As such, personalised targeted immunotherapy could be adapted. Further investigations required to map the immune evasion machinery for each type of cancer and design the best targets for personalised immunotherapy of human malignancies. <h3>Disclosure Information</h3> <b>V.V. Sumbayev:</b> None. <b>I. Yasinska:</b> None. <b>S. Schlichtner:</b> None. <b>G.S. Lall:</b> None. <b>E. Fasler-Kan:</b> None. <b>B.F. Gibbs:</b> None.

Abstract 5727: Using ChIP-seq to identify genes regulated by RelA or RelB that support ovarian cancer tumor-initiating cell (TIC) characteristics

Abstract Ovarian cancer is the most lethal gynecologic malignancy in the US. Although high-grade serous ovarian cancer (HGSOC) patients initially respond to chemotherapy, over 70% relapse in two years. This paradigm can be understood through ovarian cancer’s pronounced heterogeneity whereby a majority of cancer cells are highly proliferative and chemosensitive and a minority of cells, termed tumor-initiating cells (TICs), are relatively quiescent and chemoresistant stem-like cells. Both are presumed to be important for tumor repopulation. Our lab previously demonstrated that TICs exhibit an upregulation of stem cell genes and NF-κB signaling. RNA sequencing of cells grown in TIC enhancing, non-adherent conditions shows significantly increased expression of NF-kB genes NFKB2, RELA, and RELB, as well as stem cell genes SOX2 and ALDH1A2. In this study, we are investigating the mechanism through which the NF-κB transcription factors RelA and RelB support TICs to promote relapse in ovarian cancer. RNA sequencing of shRNA knockdowns of RELA or RELB from cells grown in TIC conditions shows that RELA knockdown impacts 1415 unique genes, RELB knockdown impacts 2016 unique genes, and RELA or RELB knockdown decreases expression of 1912 shared genes. Gene ontology analysis suggests RELA regulates genes in growth and differentiation pathways while RELB regulates genes in metabolic pathways. Specifically, RELA knockdown significantly decreased expression of NF-kB pathway genes (RELA, RELB, NFKB2) as well as stem cell genes CD117 and ALDH1A2. RELB knockdown significantly decreased expression of NF-kB pathway genes (RELA, RELB) as well as stem cell genes CD117, CD133, ALDH1A1, and ALDH1A2. To expand on these findings we performed ChIP-sequencing of RelA and RelB in OV90 cells cultured in TIC enhancing or adherent monolayer conditions. Our results show that both RelA and RelB bind at promoter sites for NFKBIA and NUAK1 in both conditions. In monolayer cultures RelA uniquely binds 14 different genes, including lncRNAs, miRNAs, and Cyclin L1, important in G0-G1 cell cycle progression. In TIC conditions, RelB uniquely binds 16 different genes, including lncRNAs, miRNAs, and WNT10A, important in stem cell self-renewal. Experiments are underway to validate our top hits in ovarian cancer, using siRNA knockdowns to corroborate the genes and pathways through which RelB supports self-renewal and RelA supports cell cycle progression. The ultimate goal of this project is to identify downstream pathways regulated by NF-kB that can be targeted to overcome relapse in HGSOC. Citation Format: Emily M. Mu, Samuel F. Gilbert, Mikella Robinson, Jacqueline Lara, Omar Lujano-Olazaba, Christina M. Annunziata, Carrie D. House. Using ChIP-seq to identify genes regulated by RelA or RelB that support ovarian cancer tumor-initiating cell (TIC) characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5727.

ROS-induced cell cycle arrest as a mechanism of resistance in polyaneuploid cancer cells (PACCs)

Cancer is responsible for the deaths of millions of people worldwide each year. Once metastasized, the disease is incurable and shows resistance to all anti-cancer therapies. The already-elevated level of reactive oxygen species (ROS) in cancer cells is further increased by therapies. The oxidative stress activates the DNA damage response (DDR) and the stressed cancer cell moves towards cell cycle arrest. Once arrested, the majority of cancer cells will undergo programmed cell death in the form of apoptosis. If the cancer cell is able to exit the cell cycle prior to cell division and enter a protected G0 state, it is able to withstand and survive therapy as a polyaneuploid cancer cell (PACC) and eventually seed resistant tumor growth.

Clinical Candidates Targeting the ATR–CHK1–WEE1 Axis in Cancer

Simple SummarySelective killing of cancer cells is privileged mainstream in cancer treatment and targeted therapy represents the new tool with a potential to pursue this aim. It can also aid to overcome resistance of conventional chemo- or radio-therapy. Common mutations of cancer cells (defective G1 control) favor inhibiting intra-S and G2/M-checkpoints, which are regulated by ATR–CHK1–WEE1 pathway. The ATR–CHK1–WEE1 axis has produced several clinical candidates currently undergoing clinical trials in phase II. Clinical results from randomized trials by ATR and WEE1 inhibitors warrant ongoing clinical trials in phase III.Selective killing of cancer cells while sparing healthy ones is the principle of the perfect cancer treatment and the primary aim of many oncologists, molecular biologists, and medicinal chemists. To achieve this goal, it is crucial to understand the molecular mechanisms that distinguish cancer cells from healthy ones. Accordingly, several clinical candidates that use particular mutations in cell-cycle progressions have been developed to kill cancer cells. As the majority of cancer cells have defects in G1 control, targeting the subsequent intra‑S or G2/M checkpoints has also been extensively pursued. This review focuses on clinical candidates that target the kinases involved in intra‑S and G2/M checkpoints, namely, ATR, CHK1, and WEE1 inhibitors. It provides insight into their current status and future perspectives for anticancer treatment. Overall, even though CHK1 inhibitors are still far from clinical establishment, promising accomplishments with ATR and WEE1 inhibitors in phase II trials present a positive outlook for patient survival.

Doxorubicin sensitizes cancer cells to Smac mimetic via synergistic activation of the CYLD/RIPK1/FADD/caspase-8-dependent apoptosis

Smac/Diablo is a pro-apoptotic protein via interaction with inhibitors of apoptosis proteins (IAPs) to relieve their inhibition of caspases. Smac mimetic compounds (also known as antagonists of IAPs) mimic the function of Smac/Diablo and sensitize cancer cells to TNF-induced apoptosis. However, the majority of cancer cells are resistant to Smac mimetic alone. Doxorubicin is a widely used chemotherapeutic drug and causes adverse effect of cardiotoxicity in many patients. Therefore, it is important to find strategies of combined chemotherapy to increase chemosensitivity and reduce the adverse effects. Here, we report that doxorubicin synergizes with Smac mimetic to trigger TNF-mediated apoptosis, which is mechanistically distinct from doxorubicin-induced cell death. Doxorubicin sensitizes cancer cells including human pancreatic and colorectal cancer cells to Smac mimetic treatment. The combined treatment leads to synergistic induction of TNFα to initiate apoptosis through activating NF-κB and c-Jun signaling pathways. Knockdown of caspase-8 or knockout of FADD significantly blocked apoptosis synergistically induced by Smac mimetic and doxorubicin, but had no effect on cell death caused by doxorubicin alone. Moreover, Smac mimetic and doxorubicin-induced apoptosis requires receptor-interacting protein kinase 1 (RIPK1) and its deubiquitinating enzyme cylindromatosis (CYLD), not A20. These in vitro findings demonstrate that combination of Smac mimetic and doxorubicin synergistically triggers apoptosis through the TNF/CYLD/RIPK1/FADD/caspase-8 signaling pathway. Importantly, the combined treatment induced in vivo synergistic anti-tumor effects in the xenograft tumor model. Thus, the combined therapy using Smac mimetic and doxorubicin presents a promising apoptosis-inducing strategy with great potential for the development of anti-cancer therapy.

Carnitine palmitoyltransferase 1 A expression profile in canine mammary tumors

Genetic alterations and/or epigenetic modifications occur frequently in the majority of cancer cells. In addition to playing a crucial role as promoters of tumorigenesis, these processes can also generate metabolic pathways that are different from those in normal cells. Besides the Warburg effect, an alteration in lipid metabolism is also found in cancer cells. Thus, elucidation of the regulators involved in this metabolic reprogramming might provide tools for diagnosis, prognosis, and ultimately treatment of canine mammary tumours (CMTs) in particular. One such regulator is carnitine palmitoyltransferase 1A (CPT1A), which is involved in transportation of long-chain fatty acids into the mitochondrial matrix for beta-oxidation, thereby providing an alternative pathway for the generation of energy for tumour growth and development. In this study, the canine cell lines MDCK, CMT-U309, CMT-U27, and P114 were used as in vitro models for western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. Furthermore, western blot and immunohistochemistry were carried out to evaluate CPT1A protein expression in the CMT specimens. The CPT1A protein and mRNA expression levels were increased in the CMT cell lines relative to their levels in normal epithelial cells. Moreover, increased CPT1A expression levels were found in the CMT tissues, being inversely correlated with the tumour differentiation grade. However, additional studies are required to further specify the role of CPT1A in CMTs.

Developing Enzyme‐Responsive Nanomedicine for Inhibition of hTERT Mitochondrial Translocation

AbstractAlthough considerable efforts have been made to increase the sensitivity of anticancer agents, few strategies have been found to show general effects. Human telomerase reverse transcriptase (hTERT) is overexpressed in the majority of cancer cells. hTERT has been demonstrated to translocate to mitochondria and promote cell survival under cell stress, leading to significant resistance to chemotherapeutics in various cancer cells. Thus, inhibition of hTERT translocation from nuclei to mitochondria may be developed as an effective and general way to improve drug sensitivity. Herein, leptomycin B (LMB, a nuclear export inhibitor of hTERT) and doxorubicin are delivered into cancer cells by a mesoporous silica nanocarrier. This straightforward design can inhibit hTERT translocation and improve drug chemosensitivity in different types of cancer cells. Mechanism studies reveal that hTERT movement inhibition perturbs the protection of hTERT to mitochondria, exacerbating mitochondrial dysfunction and nuclear DNA damage. This work provides an effective method for increasing drug sensitivity based on telomerase location in cancer cells.

Alternative Lengthening of Telomeres (ALT) in Tumors and Pluripotent Stem Cells.

A telomere consists of repeated DNA sequences (TTAGGG)n as part of a nucleoprotein structure at the end of the linear chromosome, and their progressive shortening induces DNA damage response (DDR) that triggers cellular senescence. The telomere can be maintained by telomerase activity (TA) in the majority of cancer cells (particularly cancer stem cells) and pluripotent stem cells (PSCs), which exhibit unlimited self-proliferation. However, some cells, such as telomerase-deficient cancer cells, can add telomeric repeats by an alternative lengthening of the telomeres (ALT) pathway, showing telomere length heterogeneity. In this review, we focus on the mechanisms of the ALT pathway and potential clinical implications. We also discuss the characteristics of telomeres in PSCs, thereby shedding light on the therapeutic significance of telomere length regulation in age-related diseases and regenerative medicine.

Sensitive detection of telomerase activity in cells using a DNA-based fluorescence resonance energy transfer nanoprobe

Telomerase activity is inhibited in normal somatic cells but abnormally high in the majority of cancer cells. Maintenance of active telomerase in cancer cells promotes proliferation and immortalization. With the difference in telomerase activity between cancer and normal cells in mind, we designed a nanoprobe based on quantum dot (QD) and fluorescence resonance energy transfer (FRET). The nanoprobe consisted of a specific sequence of DNA with the two ends labeled with QD as a fluorescent donor and Alexa488 as a fluorescent acceptor, respectively. FRET signal tracking was performed by adjusting the distance between donors and acceptors, and changes in the FRET signal shown to be related to telomerase activity. Incubation of cells with the nanoprobe facilitated sensing of intracellular telomerase activity, and consequent discrimination between normal and cancer cells. Our novel DNA nanoprobe based on QD-FRET achieved sensitive detection of telomerase in cells up to a detection limit of one cell, and quantitative detection of telomerase activity in different numbers of cells. The nanoprobe generated in this study is expected to allow dynamically monitoring of the changes in telomerase activity in cells under treatment with drugs, providing a potential basis for early diagnosis and management of cancer.

PITX1 protein interacts with ZCCHC10 to regulate hTERT mRNA transcription.

Telomerase is a ribonucleoprotein ribonucleic enzyme that is essential for cellular immortalization via elongation of telomere repeat sequences at the end of chromosomes. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase holoenzyme, is a key regulator of telomerase activity. Telomerase activity, which has been detected in the majority of cancer cells, is accompanied by hTERT expression, suggesting that this enzyme activity contributes to an unlimited replication potential of cancer cells via regulation of telomere length. Thus, hTERT is an attractive target for cancer-specific treatments. We previously reported that pared-like homeodomain 1 (PITX1) is a negative regulator of hTERT through direct binding to the hTERT promoter. However, the mechanism by which the function of PITX1 contributes to transcriptional silencing of the hTERT gene remains to be clarified. Here, we show that PITX1 and zinc finger CCHC-type containing 10 (ZCCHC10) proteins cooperate to facilitate the transcriptional regulation of the hTERT gene by functional studies via FLAG pull-down assay. Co-expression of PITX1 and ZCCHC10 resulted in inhibition of hTERT transcription, in melanoma cell lines, whereas mutate-deletion of homeodomain in PITX1 that interact with ZCCHC10 did not induce similar phenotypes. In addition, ZCCHC10 expression levels showed marked decrease in the majority of melanoma cell lines and tissues. Taken together, these results suggest that ZCCHC10-PITX1 complex is the functional unit that suppresses hTERT transcription, and may play a crucial role as a novel tumor suppressor complex.

Isolation and detection of exosomes via AAO membrane and QCM measurement

Increasing evidence suggests that exosomes are secreted by the majority of cancer cells and present in myriad biological fluids. They have been recognized as a potential new class of biomarkers for disease prognosis and diagnosis. The isolation and quantification of exosomes are crucial in facilitating exosome research. This paper presents a filtration method to isolate exosomes via anodic aluminum oxide (AAO) membrane and to quantify them using a quartz crystal microbalance (QCM). The filtration chip was designed to have two nano-porous AAO membranes sandwiched in between PDMS layers. In this study, fetal bovine serum (FBS) was adopted as the analyte for exosomal bioassays. The experimental results show that AAO membrane has a hydrophilic surface able to sieve exosomes by gravity. The spectrophotometric measurement and the result of gel electrophoresis confirmed that most of proteins in FBS were removed, leaving pure and undamaged exosomes for further analyses. Then, QCM measurement of the filtered FBS solution can give a quantitative analysis of exosome concentration. The relationship between the exosome concentration and its corresponding QCM's frequency shift is linear and monotonic. QCM is still sensitive to measure low concentration of exosomes.

Breast cancer in patients with germline TP53 pathogenic variants have typical tumour characteristics: the Cohort study of TP53 carrier early onset breast cancer (COPE study).

Germline TP53 pathogenic variants are rare but associated with a high risk of cancer; they are often identified in the context of clinically diagnosed Li–Fraumeni syndrome predisposing to a range of young onset cancers including sarcomas and breast cancer. The study aim was to conduct a detailed morphological review and immuno‐phenotyping of breast cancer arising in carriers of a germline TP53 pathogenic variant. We compared breast cancers from five defined groups: (1) TP53 carriers with breast cancer (n = 59), (2) early onset HER2‐amplified breast cancer, no germline pathogenic variant in BRCA1/2 or TP53 (n = 55), (3) BRCA1 pathogenic variant carriers (n = 60); (4) BRCA2 pathogenic variant carriers (n = 61) and (5) young onset breast cancer with no known germline pathogenic variant (n = 98). Pathologists assessed a pre‐agreed set of morphological characteristics using light microscopy. Immunohistochemistry (IHC) for HER2, ER, PR, p53, integrin alpha v beta 6 (αvβ6) integrin, α‐smooth muscle actin (α‐SMA) and pSMAD2/3 was performed on tissue microarrays of invasive carcinoma. We confirmed a previously reported high prevalence of HER2‐amplified, ductal no special type invasive breast carcinoma amongst known TP53 germline pathogenic variant carriers 20 of 36 (56%). Furthermore we observed a high frequency of densely sclerotic tumour stroma in cancers from TP53 carriers (29/36, 80.6%) when compared with non‐carriers, 50.9% (28/55), 34.7% (50/144), 41.4% (65/157), 43.8% (95/217) in groups 2–5 respectively. The majority of germline TP53 gene carrier breast tumours had a high intensity of integrin αvβ6, α‐SMA and pSMAD2/3 expression in the majority of cancer cells. In conclusion, aggressive HER2 positive breast cancers with densely sclerotic stroma are common in germline TP53 carriers. High levels of αvβ6 integrin, α‐SMA and pSMAD2/3 expression suggest that the dense stromal phenotype may be driven by upregulated transforming growth factor beta signalling.

Integrator is a key component of human telomerase RNA biogenesis

Telomeres are special DNA-protein structures that are located at the ends of linear eukaryotic chromosomes. The telomere length determines the proliferation potential of cells. Telomerase is a key component of the telomere length maintenance system. While telomerase is inactive in the majority of somatic cells, its activity determines the clonogenic potential of stem cells as a resource for tissue and organism regeneration. Reactivation of telomerase occurs during the process of immortalization in the majority of cancer cells. Telomerase is a ribonucleoprotein that contains telomerase reverse transcriptase and telomerase RNA components. The RNA processing mechanism of telomerase involves exosome trimming or degradation of the primary precursor. Recent data provide evidence that the competition between the processing and decay of telomerase RNA may regulate the amount of RNA at the physiological level. We show that termination of human telomerase RNA transcription is dependent on its promoter, which engages with the multisubunit complex Integrator to interact with RNA polymerase II and terminate transcription of the human telomerase RNA gene followed by further processing.

Real-Time Determination of the Cell-Cycle Position of Individual Cells within Live Tumors Using FUCCI Cell-Cycle Imaging.

Most cytotoxic agents have limited efficacy for solid cancers. Cell-cycle phase analysis at the single-cell level in solid tumors has shown that the majority of cancer cells in tumors is not cycling and is therefore resistant to cytotoxic chemotherapy. Intravital cell-cycle imaging within tumors demonstrated the cell-cycle position and distribution of cancer cells within a tumor, and cell-cycle dynamics during chemotherapy. Understanding cell-cycle dynamics within tumors should provide important insights into novel treatment strategies.

Impact of COX2 Inhibitor for Regulation of PD-L1 Expression in Non-small Cell Lung Cancer.

There is no clear evidence in the literature regarding the regulation of programmed cell death-ligand 1 (PD-L1) expression by cyclo-oxygenase-2 (COX2). In this study, whether PD-L1 expression was regulated by COX2 activity was examined in vitro. Resected lung cancer specimens were analyzed for PD-L1 and COX2 expression by immunohistochemical analysis. Next, co-localization of PD-L1 and COX2 expression was analyzed by double-fluorescence staining. Lastly, the effect of COX2 inhibition on the expression of PD-L1 was examined using lung cancer cell lines. PD-L1 expression was significantly correlated with COX2 expression in the resected specimens. The majority of cancer cells that expressed PD-L1 also co-expressed COX2. However, treatment of lung cancer cell lines with a COX2 inhibitor had no impact on PD-L1 expression. Our results suggest that COX2 inhibition might have no effect on the usage of immune checkpoint inhibitors in lung cancer treatment.