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Fluorescence microscopy time series of doxorubicin or liposomal doxorubicin uptake into human tumor cell lines MDA-MB-231. a Cells were treated with 12 µM doxorubicin for 1, 2, 4, and 8 h as indicated. b Cells were treated with 12 µM liposomal formulated doxorubicin for 1, 2, 4, and 8 h as indicated. Control cells remained
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![Fig. 3 Immunohistochemical detection of cisplatin Pt-[GpG] DNA adducts...](publication/339688267/figure/fig3/AS:898397913481220@1591206162837/mmunohistochemical-detection-of-cisplatin-Pt-GpG-DNA-adducts-in-paraffin-sections-of_Q320.jpg)
![Fig. 4 Immunohistochemical detection of cisplatin Pt-[GpG] DNA adducts...](publication/339688267/figure/fig4/AS:898397913501701@1591206162988/mmunohistochemical-detection-of-cisplatin-Pt-GpG-DNA-adducts-in-paraffin-sections-of_Q320.jpg)
A major limitation in the pharmacological treatment of clinically detectable primary cancers and their metastases is their limited accessibility to anti-cancer drugs (cytostatics, inhibitory antibodies, small-molecule inhibitors) critically impairing therapeutic efficacies. Investigations on the tissue distribution of such drugs are rare and have o...
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Context 1
... capture the uptake of doxorubicin or liposomal doxorubicin, respectively, into the nucleus of cells, we carried out a time series experiment (Fig. 2). For doxorubicin, maximal fluorescence intensity was reached after incubation of MDA-MB-231 cells for 4 h with no further increase at the 8 h time point (Fig. 2a). Nuclear uptake of liposomal formulated doxorubicin was somewhat slower with lower fluorescence intensity and showed only a small increase at the 8 h time point (Fig. ...
Context 2
... capture the uptake of doxorubicin or liposomal doxorubicin, respectively, into the nucleus of cells, we carried out a time series experiment (Fig. 2). For doxorubicin, maximal fluorescence intensity was reached after incubation of MDA-MB-231 cells for 4 h with no further increase at the 8 h time point (Fig. 2a). Nuclear uptake of liposomal formulated doxorubicin was somewhat slower with lower fluorescence intensity and showed only a small increase at the 8 h time point (Fig. ...
Context 3
... experiment (Fig. 2). For doxorubicin, maximal fluorescence intensity was reached after incubation of MDA-MB-231 cells for 4 h with no further increase at the 8 h time point (Fig. 2a). Nuclear uptake of liposomal formulated doxorubicin was somewhat slower with lower fluorescence intensity and showed only a small increase at the 8 h time point (Fig. ...
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The pandemic of Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome 2 coronavirus (SARS-CoV-2) continues to be a global health crisis. Fundamental studies at genome, transcriptome, proteome, and interactome levels have revealed many viral and host targets for therapeutic interventions. Hundreds of antibodies for trea...
Citations
... Our results clearly indicate that the energy providing function of mitochondria is of greater importance for the intraperitoneal The results of the survival analysis suggest that the function of a suicidal weapon store is of lesser importance for metastasis formation of ovarian cancer cells than the function of energy provision for cellular activities in which a high mitochondrial content is associated with poorer survival. This observation may be explained in part by the occurrence of high interstitial fluid pressure within solid tumours, which shields the majority of cancer cells from attack by chemotherapeutic drugs because they cannot enter the mass of the primary tumour as they are too far away from circulation [28,29]. Hence the functions of a suicidal weapon store are not needed in cancer cells further away from functioning exchange blood vessels. ...
Most cancer patients ultimately die from the consequences of distant metastases. As metastasis formation consumes energy mitochondria play an important role during this process as they are the most important cellular organelle to synthesise the energy rich substrate ATP, which provides the necessary energy to enable distant metastasis formation. However, mitochondria are also important for the execution of apoptosis, a process which limits metastasis formation. We therefore wanted to investigate the mitochondrial content in ovarian cancer cells and link its presence to the patient’s prognosis in order to analyse which of the two opposing functions of mitochondria dominates during the malignant progression of ovarian cancer. Monoclonal antibodies directed against different mitochondrial specific proteins, namely heat shock proteins 60 (HSP60), fumarase and succinic dehydrogenase, were used in immunohistochemistry in preliminary experiments to identify the antibody most suited to detect mitochondria in ovarian cancer cells in clinical tissue samples. The clearest staining pattern, which even delineated individual mitochondria, was seen with the anti-HSP60 antibody, which was used for the subsequent clinical study staining primary ovarian cancers (n = 155), borderline tumours (n = 24) and recurrent ovarian cancers (n = 26). The staining results were semi-quantitatively scored into three groups according to their mitochondrial content: low (n = 26), intermediate (n = 50) and high (n = 84). Survival analysis showed that high mitochondrial content correlated with a statistically significant overall reduced survival rate In addition to the clinical tissue samples, mitochondrial content was analysed in ovarian cancer cells grown in vitro (cell lines: OVCAR8, SKOV3, OVCAR3 and COV644) and in vivo in severe combined immunodeficiency (SCID) mice.
In in vivo grown SKOV3 and OVCAR8 cells, the number of mitochondria positive cells was markedly down-regulated compared to the in vitro grown cells indicating that mitochondrial number is subject to regulatory processes. As high mitochondrial content is associated with a poor prognosis, the provision of high energy substrates by the mitochondria seems to be more important for metastasis formation than the inhibition of apoptotic cell death, which is also mediated by mitochondria. In vivo and in vitro grown human ovarian cancer cells showed that the mitochondrial content is highly adaptable to the growth condition of the cancer cells.
... Simultaneously, cisplatin has substantial side effects including ototoxicity, bone marrow suppression and neurotoxicity, with large individual variability in the occurrence of these side effects during different chemotherapy cycles. Several approaches for detecting cisplatin have been developed, such as colorimetric [2], inductively coupled plasma mass spectrometry (ICP-MS) [3], high performance liquid chromatography (HPLC) [4] and improved schemes based on above three methods [5] [6]. However, these approaches are high-interference, expensive with advanced analytical instruments, and require extensive sample pre-treatment, which cannot meet the demand for clinical sample detection. ...
Cisplatin, one of the most widely used clinical medications in the field of cancer chemotherapy, which is an indispensable drug type in traditional chemotherapy. Due to the significant toxicity and side effects of typical chemotherapy medications, precise drug concentration monitoring becomes the key. However, because of individual variances, various individuals’ metabolic capacities to medications vary, resulting in differences in actual blood drug concentration in the body under the same dosage. Therefore, it is imperative to create a new low-cost and rapid detection method for the concentration of serum active cisplatin, which is crucial to the chemotherapy effect and quality of life for cancer patients. In this work, based on the chemically cross-linked reaction between active cisplatin and DNA bases, as well as the high sensitivity of Fluorescence Resonance Energy Transfer (FRET), we proposed a new FRET-based method for detecting serum active cisplatin concentration. We constructed a fluorescent molecular probe with a dumbbell shape consisting of graphene quantum dots (GQDs) modified with carboxyl, gold nanoparticles (AuNPs), and a single-stranded DNA (ssDNA) sequence. The results reveal that the molecular probe has a strong linear correlation across the active cisplatin concentration range of 52-832 μM. This method can accomplish low-cost, rapid and simple detection of active cisplatin concentration, and further advancements in this work can give a decision-making basis for tailored and accurate cancer treatment.
... Examination of tumor tissue also revealed the accumulation of brown pigment, DOX, in mitotic division figures and cell nuclei in mice treated with (DOX+SFN)-lip. It has previously been shown that DOX may be detected in cell nuclei in H&E sections due to its stable incorporation into DNA, in contrast to free DOX, which may be washed out during tissue processing [33]. These findings are in agreement with in vitro microscopic observation of enhanced accumulation of DOX after incubation of cells with (DOX+SFN)-lip. ...
Female breast cancer is the most deadly cancer in women worldwide. The triple-negative breast cancer subtype therapies, due to the lack of specific drug targets, are still based on systemic chemotherapy with doxorubicin, which is burdened with severe adverse effects. To enhance therapeutic success and protect against systemic toxicity, drug carriers or combination therapy are being developed. Thus, an innovative liposomal formulation containing doxorubicin and the main nutraceutical, sulforaphane, has been developed. The anticancer efficacy and safety of the proposed liposomal formulation was evaluated in vivo, in a 4T1 mouse model of triple-negative breast cancer, and the mechanism of action was determined in vitro, using triple-negative breast cancer MDA-MB-231 and non-tumorigenic breast MCF-10A cell line. The elaborated drug carriers were shown to efficiently deliver both compounds into the cancer cell and direct doxorubicin to the cell nucleus. Incorporation of sulforaphane resulted in a twofold inhibition of tumor growth and the potential of up to a fourfold reduction in doxorubicin concentration due to the synergistic interaction between the two compounds. Sulforaphane was shown to increase the accumulation of doxorubicin in the nuclei of cancer cells, accompanied by inhibition of mitosis, without affecting the reactive oxygen species status of the cell. In normal cells, an antagonistic effect resulting in less cytotoxicity was observed. In vivo results showed that sulforaphane incorporation yielded not only cardioprotective, but also nephro- and hepatoprotective effects. The results of the research revealed the prospects of applying sulforaphane as a component of liposomal doxorubicin in triple-negative breast cancer chemotherapy.
... Preparation of cancer cells as formalin-fixed paraffin-embedded (FFPE) samples was carried out as previously described [35]. Briefly, cancer cells were collected from culture flasks, fixed in formalin and next embedded into agar pellets. ...
Background
YKL-40, also known as non-enzymatic chitinase-3 like-protein-1 (CHI3L1), is a glycoprotein expressed and secreted mainly by inflammatory cells and tumor cells. Accordingly, several studies demonstrated elevated YKL-40 serum levels in cancer patients and found YKL-40 to be correlated with a poor prognosis and disease severity in some tumor entities. YKL-40 was suggested to be involved in angiogenesis and extracellular matrix remodeling. As yet, however, its precise biological function remains elusive.
Methods
As YKL-40 protein expression has only been investigated in few malignancies, we employed immunohistochemical detection in a large multi-tumor tissue microarray consisting of 2,310 samples from 72 different tumor entities. In addition, YKL-40 protein expression was determined in primary mouse xenograft tumors derived from human cancer cell lines.
Results
YKL-40 could be detected in almost all cancer entities and was differently expressed depending on tumor stage and subtype (e.g., thyroid cancer, colorectal cancer, gastric cancer and ovarian cancer). While YKL-40 was absent in in vitro grown human cancer cell lines, YKL-40 expression was upregulated in xenograft tumor tissues in vivo.
Conclusions
These data provide new insights into YKL-40 expression at the protein level in various tumor entities and its regulation in tumor models. Our data suggest that upregulation of YKL-40 expression is a common feature in vivo and is finely regulated by tumor cell-microenvironment interactions.
... Then, fixed cells were washed twice with PBS. Then, cells were embedded in agar as previously described [11]. Briefly, the cell pellets were resuspended with 300 µL of 2% Difco TM Noble Agar (Becton, Dickinson, Sparks, MD, USA), which was preheated up to a temperature of 55°C. ...
Ovarian cancer (OvCa) disseminates primarily intraperitoneally. Detached tumor cell aggregates (spheroids) from the primary tumor are regarded as “metastatic units” that exhibit a low sensitivity to classical chemotherapy, probably due to their unique molecular characteristics. We have analyzed the cellular composition of ascites from OvCa patients, using flow cytometry, and studied their behavior in vitro and in vivo. We conclude that ascites‐derived cultured cells from OvCa patients give rise to two subpopulations: adherent cells (ADs) and non‐adherent cells (NADs). Here, we found that the AD population includes mainly CD90+ cells with highly proliferative rates in vitro but no tumorigenic potential in vivo, whereas the NAD population contains principally tumor cell spheroids (EpCAM+/CD24+) with low proliferative potential in vitro. Enriched tumor cell spheroids from the ascites of high‐grade serous OvCA (HGSOC) patients, obtained using cell strainers, were highly tumorigenic in vivo and their metastatic spread pattern precisely resembled the tumor dissemination pattern found in the corresponding patients. Comparative transcriptome analyses from ascites‐derived tumor cell spheroids (n=10) versus tumor samples from different metastatic sites (n=30) revealed upregulation of genes involved in chemoresistance (TGM1, HSPAs, MT1s), cell adhesion and cell‐barrier integrity (PKP3, CLDNs, PPL), and the oxidative phosphorylation (OXPHOS) process. Mitochondrial markers (mass and membrane potential) showed a reduced mitochondrial function in tumoroids from tumor tissue compared with ascites‐derived tumor spheroids in flow cytometry analysis. Interestingly, response to OXPHOS inhibition by metformin and IACS010759 in tumor spheroids correlated with the extent of mitochondrial membrane potential measured by fluorescence‐activated cell sorting (FACS). Our data contribute to a better understanding of the biology of ovarian cancer spheroids and identify the OXPHOS pathway as new potential treatment option in advanced ovarian cancer.
... Cisplatin is a well-known chemotherapeutic agent which belongs to the DNA alkylating family [44], used for the treatment of numerous human cancers, including bladder, head, neck, lung, ovarian and testicular cancers [45]. Cisplatin binds to nucleophilic groups in DNA, introducing intra-and interstrand DNA cross-links which lead to growth inhibition and apoptosis [46,47]. Cisplatin is known for its cytotoxic and genotoxic effects, both in vitro and in vivo, mainly due to the production of radical oxygen species (ROS) [48,49]. ...
Phospholipase A2 Bothropstoxin-I (PLA2 BthTX-I) is a myotoxic Lys49-PLA2 from Bothrops jararacussu snake venom. In order to evaluate the DNA damage caused by BthTX-I, we used the Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster and Comet assay in HUVEC and DU-145 cells. For SMART, different concentrations of BthTX-I (6.72 to 430 μg/mL) were used and no significant changes in the survival rate were observed. Significant frequency of mutant spots was observed for the ST cross at the highest concentration of BthTX-I due to recombinogenic activity. In the HB cross, BthTX-I increased the number of mutant spots at intermediate concentrations, being 53.75 μg/mL highly mutagenic and 107.5 μg/mL predominantly recombinogenic. The highest concentrations were neither mutagenic nor recombinogenic, which could indicate cytotoxicity in the wing cells of D. melanogaster. In vitro, all BthTX-I concentrations (1 to 50 μg/mL) induced decrease in HUVEC cell viability, as well as in DU-145 cells at concentrations of 10, 25, and 50 μg/mL. The comet assay showed that in HUVEC and DU-145 cells, all BthTX-I concentrations promoted increase of DNA damage. Further studies should be performed to elucidate the mechanism of action of PLA2 BthTX-I and its possible use in therapeutic strategies against cancer.
... Notably, this is somewhat contradictory to other observations that suggest impaired tissue penetration already of single molecules, e.g., biologicals or even small molecule cytostatics (see e.g. 43 ). In fact, this may indicate the need for a delicate balance between cell uptake efficacy, which has to be sufficiently high for biological effects and adequately low for allowing deeper tissue penetration without interference only with the first cell layers hit after extravasation into their target tissue. ...
Therapeutic gene silencing by RNA interference relies on the safe and efficient in vivo delivery of small interfering RNAs (siRNAs). Polyethylenimines are among the most studied cationic polymers for gene delivery. For several reasons including superior tolerability, small linear PEIs would be preferable over branched PEIs, but they show poor siRNA complexation. Their chemical modification for siRNA formulation has not been extensively explored so far.
We generated a set of small linear PEIs bearing tyrosine modifications (LPxY), leading to substantially enhanced siRNA delivery and knockdown efficacy in vitro in various cell lines, including hard-to-transfect cells. The tyrosine-modified linear 10 kDa PEI (LP10Y) is particularly powerful, associated with favorable physicochemical properties and very high biocompatibility. Systemically administered LP10Y/siRNA complexes reveal antitumor effects in mouse xenograft and patient-derived xenograft (PDX) models, and their direct application into the brain achieves therapeutic inhibition of orthotopic glioma xenografts. LP10Y is particularly interesting for therapeutic siRNA delivery.
... Preparation of cancer cells as formalin-fixed paraffin-embedded (FFPE) samples was carried out as previously described [49]. Briefly, cancer cells were collected from culture flasks, fixed in formalin and then embedded into agar pellets. ...
Simple Summary
The metabolic protein TXNIP plays a crucial role in various cellular processes. Abnormal TXNIP levels are notable, e.g., in type II diabetes, cardiovascular diseases, and tumors. Using immunohistochemical staining for TXNIP in different tumor entities, we give new insights of TXNIP expression on the protein level. In human tumors, staining intensity inversely correlated with aggressiveness of the tumor entity. In contrast, human tumor cell lines grown in mice (xenografts), consistently revealed no staining. Hence, loss of TXNIP suggests a critical role for the development of tumors in xenografts. Furthermore, we investigated TXNIP staining of immunocompetent cells in the proximity of the xenograft tumor tissue. Our findings demonstrate that TXNIP downregulation is a common feature in human tumor xenograft models. Subsequently, TXNIP expression might be used to monitor the functional state of tumor-infiltrating leukocytes in tissue sections and may help to predict response to modern immune therapy.
Abstract
Thioredoxin interacting protein (TXNIP) is a metabolic protein critically involved in redox homeostasis and has been proposed as a tumor suppressor gene in a variety of malignancies. Accordingly, TXNIP is downregulated in breast, bladder, and gastric cancer and in tumor transplant models TXNIP overexpression inhibits growth and metastasis. As TXNIP protein expression has only been investigated in few malignancies, we employed immunohistochemical detection in a large multi-tumor tissue microarray consisting of 2,824 samples from 94 different tumor entities. In general, TXNIP protein was present only in a small proportion of primary tumor samples and in these cases was differently expressed depending on tumor stage and subtype (e.g., renal cell carcinoma, thyroid cancer, breast cancer, and ductal pancreatic cancer). Further, TXNIP protein expression was determined in primary mouse xenograft tumors derived from human cancer cell lines and was immunohistochemically absent in all xenograft tumors investigated. Intriguingly, TXNIP expression became gradually lower in the proximity of the primary tumor tissue and was absent in leukocytes directly adjacent to tumor tissue. In conclusion, these findings suggest that TXNIP downregulation is as a common feature in human tumor xenograft models and that intra-tumoral leukocytes down-regulate TXNIP. Hence TXNIP expression might be used to monitor the functional state of tumor-infiltrating leukocytes in tissue sections.
Background: Ovarian cancer (OvCa) cells disseminate primarily intraperitoneally. Here, detached tumor cell aggregates (spheroids) from the primary tumor are generally regarded as “metastatic units”, which exhibit a survival benefit, probably due to the protective microenvironment and their unique molecular characteristics. Hence, current therapeutic concepts such as classical chemotherapy are not sufficient preventing growth and spread of OvCa spheroids.
Methods: In the current study we analyzed the cellular composition of ascites from ovarian cancer patients using flow cytometry and the tumorigenic potential of the different subpopulations in an intraperitoneal mouse model. Comparative transcriptome analyses (RNAseq) from ascites-derived tumor cells spheroids (n=10) vs. tumor samples from different metastatic sites (n=30) were further performed in order to identify key molecular players responsible for the special cellular characteristics of OvCa spheroids.
Results: In vitro culture of ascites-derived cells gave rise to two different subpopulations: an adherent cell population (ADs) including mainly CD90+ cells with highly proliferative rates in vitro but no tumorigenic potential in vivo, and a non-adherent cell population (NADs) containing principally EpCAM+/CD24+ cells with low proliferative potential in vitro. NADs included cell aggregates and single cells, the first showing a high content (> 80%) of tumor cells (EpCAM+/CD24+). Enriched tumor cell spheroids from the ascites using cell strainers showed higher tumorigenic potential in vivo in comparison to the original ascites-derived cell population. Interestingly, the different metastatic spread patterns observed in the mice resembled the tumor dissemination pattern found in the corresponding patients. RNAseq analyses from tumor-spheroids revealed up-regulation of genes involved in chemoresistance (TGM1, HSPAs, MT1s), cell-adhesion and cell barrier (PKP3, CLDNs, PPL) and the oxidative phosphorylation (OXPHOS) process compared to the solid tumor tissue samples. Additionally, down-regulation of extracellular matrix components and angiogenesis-related genes could be observed. Targeting OXPHOS by metformin treatment led to reduced viability of ascites-derived spheroids from OvCa patients, showing to some extent a synergistic effect with cisplatin treatment.
Conclusions: the actual study contributes to a better understanding of the biology of ovarian cancer spheroids and to the identification of new treatment opportunities in advanced ovarian cancer.
