bluuebunny/crossref_metadata_2025 huggingface dataset:
Health‐Care Technology of Badminton Sports Based on Nanotechnology
Authors: Wen Qiuning
Published: June 2022

Nanotechnology has become an important technology that cannot be ignored in high and new technology, and the application of nanomaterials is to design all aspects of life. With the more popularization of badminton, the blending of the two is also increasing. The purpose of this paper is to discuss the combination of sports health care and nanotechnology in badminton. This paper starts with the application of nanotechnology in badminton racket materials, hoping to explore the impact of nanotechnology on badminton sports health care. In this paper, 130 testers of different ages from different schools were selected for 17 weeks of badminton training. By comparing the physical fitness indicators before and after, this paper finds the impact of badminton on physical health. The experimental results show that badminton exercise training has an impact on human health and has a significant impact on human height, weight, and lung capacity.
Multigraph Aggregation Spatiotemporal Graph Convolution Network for Ride-Hailing Pick-Up Region Prediction
Authors: Li Cong , Zhang Huyin , Wang Zengkai , Wu Yonghao , Yang Fei
Published: June 2022

The prediction of pick-up regions for online ride-hailing can reduce the number of vacant vehicles on the streets, which will optimize the transportation efficiency of cities, reduce energy consumption and carbon emissions, and increase the income of online ride-hailing drivers. However, traditional studies have ignored the temporal and spatial dependencies among pick-up regions and the effects of similarity of POI attributes in different regions in modelling, making the features of the model incomplete. To address the above problems, we propose a new multigraph aggregation spatiotemporal graph convolutional network (MAST-GCN) model to predict pick-up regions for online ride-hailing. In this paper, we propose a graph aggregation method to extract the spatiotemporal aspects and preference features of spatial graphs, order graphs, and POI graphs. GCN is used on the aggregated graphs to extract spatial dimensional features from graph-structured data. The historical data are sequentially divided into temporal granularity according to the period, and convolution operations are performed on the time axis to obtain the features in the temporal dimension. The attention mechanism is used to assign different weights to features with strong periodicity and strong correlation, which effectively solves the pick-up region prediction problem. We implemented the MAST-GCN model based on the PyTorch framework, stacked with a two-layer spatiotemporal graph convolution module, where the dimension of the graph convolution is 64. We evaluate the proposed model on two real-world large scale ride-hailing datasets. The results show that our method provides significant improvements over state-of-the-art baselines.
Prostaglandin EP receptors: Targets for treatment and prevention of colorectal cancer?
Authors: Hull Mark A. , Ko Stanley C.W. , Hawcroft Gillian
Published: June 2022

The importance of the prostaglandin (PG) synthesis pathway, particularly the rate-limiting enzymatic step catalyzed by cyclooxygenase, to colorectal carcinogenesis and development of novel anticolorectal cancer therapy is well established. The predominant PG species in benign and malignant colorectal tumors is PGE2. PGE2 acts via four EP receptors termed EP1 to EP4. Recently, EP receptors have been identified as potential targets for treatment and/or prevention of colorectal cancer. This review summarizes existing knowledge of the expression and function of the EP receptor subtypes in human and rodent intestine during tumorigenic progression and describes the current literature on targeting EP receptor signaling during intestinal tumorigenesis.
Structure-based design of a selective heparanase inhibitor as an antimetastatic agent
Authors: Ishida Keisuke , Hirai Go , Murakami Koji , Teruya Takayuki , Simizu Siro , Sodeoka Mikiko , Osada Hiroyuki
Published: June 2022

Heparanase is an endo-β-d-glucuronidase that degrades heparan sulfate glycosaminoglycans in the extracellular matrix and the basement membrane and is well known to be involved in tumor cell invasion and angiogenesis. We have focused on heparanase as a target for antitumor agents, especially antimetastatic agents. (R)-3-hexadecanoyl-5-hydroxymethyltetronic acid (RK-682) was found to display an inhibitory activity against heparanase in our screening of natural sources. Because RK-682 has been reported to show inhibitory activities against several enzymes, we have tried to develop selective heparanase inhibitors using the method of rational drug design. Based on the structure of the heparanase/RK-682 complex, we speculated that selective inhibitory activity against heparanase could be acquired by arylalkylation, namely, by benzylation of the 4-position of RK-682. Among the rationally designed 4-alkyl-RK-682 derivatives, 4-benzyl-RK-682 has been found to possess a selective inhibitory activity for heparanase (IC50 for heparanase, 17 μmol/L; IC50 for other enzymes, >100 μmol/L). 4-Benzyl-RK-682 also inhibited the invasion and migration of human fibrosarcoma HT1080 cells (IC50 for invasion, 1.5 μmol/L; IC50 for migration, 3.0 μmol/L). On the other hand, RK-682 had no inhibitory effect on the invasion and migration of HT1080 cells at doses of up to 100 μmol/L.
Gefitinib reverses breast cancer resistance protein–mediated drug resistance
Authors: Yanase Kae , Tsukahara Satomi , Asada Sakiyo , Ishikawa Etsuko , Imai Yasuo , Sugimoto Yoshikazu
Published: June 2022

Breast cancer resistance protein (BCRP) is an ATP binding cassette transporter that confers resistance to a series of anticancer agents such as 7-ethyl-10-hydroxycamptothecin (SN-38), topotecan, and mitoxantrone. In this study, we evaluated the possible interaction of gefitinib, a selective epidermal growth factor receptor tyrosine kinase inhibitor, with BCRP. BCRP-transduced human epidermoid carcinoma A431 (A431/BCRP) cells acquired cellular resistance to gefitinib, suggesting that BCRP could be one of the determinants of gefitinib sensitivity in a certain sort of cells. Next, the effect of gefitinib on BCRP-mediated drug resistance was examined. Gefitinib reversed SN-38 resistance in BCRP-transduced human myelogenous leukemia K562 (K562/BCRP) or BCRP-transduced murine lymphocytic leukemia P388 (P388/BCRP) cells but not in these parental cells. In addition, gefitinib sensitized human colon cancer HT-29 cells, which endogenously express BCRP, to SN-38. Gefitinib increased intracellular accumulation of topotecan in K562/BCRP cells and suppressed ATP-dependent transport of estrone 3-sulfate, a substrate of BCRP, in membrane vesicles from K562/BCRP cells. These results suggest that gefitinib may overcome BCRP-mediated drug resistance by inhibiting the pump function of BCRP. Furthermore, P388/BCRP-transplanted mice treated with combination of irinotecan and gefitinib survived significantly longer than those treated with irinotecan alone or gefitinib alone. In conclusion, gefitinib is shown to interact with BCRP. BCRP expression in a certain sort of cells is supposed to be one of the determinants of gefitinib sensitivity. Gefitinib inhibits the transporter function of BCRP and reverses BCRP-mediated drug resistance both in vitro and in vivo.
Neuronal-associated microtubule proteins class III β-tubulin and MAP2c in neuroblastoma: Role in resistance to microtubule-targeted drugs
Authors: Don Sima , Verrills Nicole M. , Liaw Tracy Y.E. , Liu Marjorie L.M. , Norris Murray D. , Haber Michelle , Kavallaris Maria
Published: June 2022

Advanced stage neuroblastoma has a poor clinical outcome and microtubule-destabilizing agents, such as the Vinca alkaloids, are an important component in the treatment of this childhood cancer. Vinca alkaloids bind to β-tubulin on the α/β-tubulin heterodimer and disrupt microtubule dynamics, leading to cell death. To date, studies examining the contribution of microtubules and associated proteins to the efficacy of microtubule-destabilizing agents in neuroblastoma have been limited. In this study, BE(2)-C neuroblastoma cells previously selected for resistance to either vincristine (BE/VCR10) or colchicine (BE/CHCb0.2) were found to display significant decreases in neuronal-specific class III β-tubulin. Interestingly, vincristine-selected cells exhibited increased levels of polymerized tubulin that were not due to α-tubulin and class I, II, or III β-tubulin mutations. Expression levels of the microtubule-depolymerizing protein stathmin were significantly increased in BE/VCR10 cells. In contrast, levels of MAP2a and MAP2b were relatively unaltered. A marked decrease in the neuronal protein, MAP2c, was identified in the vincristine-selected cells and, to a lesser extent, in the colchicine-selected cells. This is the first report describing specific microtubule alterations in neuroblastoma cells resistant to tubulin-targeted agents. The results indicate a need to identify the factors responsible for resistance to tubulin-targeted agents in neuroblastoma so that improved and novel treatment strategies can be developed for this drug refractory disease.
Inhibition of Chk1 by the G2 DNA damage checkpoint inhibitor isogranulatimide
Authors: Jiang Xiuxian , Zhao Baoguang , Britton Robert , Lim Lynette Y. , Leong Dan , Sanghera Jasbinder S. , Zhou Bin-Bing S. , Piers Edward , Andersen Raymond J. , Roberge Michel
Published: June 2022

Inhibitors of the G2 DNA damage checkpoint can selectively sensitize cancer cells with mutated p53 to killing by DNA-damaging agents. Isogranulatimide is a G2 checkpoint inhibitor containing a unique indole/maleimide/imidazole skeleton identified in a phenotypic cell-based screen; however, the mechanism of action of isogranulatimide is unknown. Using natural and synthetic isogranulatimide analogues, we show that the imide nitrogen and a basic nitrogen at position 14 or 15 in the imidazole ring are important for checkpoint inhibition. Isogranulatimide shows structural resemblance to the aglycon of UCN-01, a potent bisindolemaleimide inhibitor of protein kinase Cβ (IC50, 0.001 μmol/L) and of the checkpoint kinase Chk1 (IC50, 0.007 μmol/L). In vitro kinase assays show that isogranulatimide inhibits Chk1 (IC50, 0.1 μmol/L) but not protein kinase Cβ. Of 13 additional protein kinases tested, isogranulatimide significantly inhibits only glycogen synthase kinase-3β (IC50, 0.5 μmol/L). We determined the crystal structure of the Chk1 catalytic domain complexed with isogranulatimide. Like UCN-01, isogranulatimide binds in the ATP-binding pocket of Chk1 and hydrogen bonds with the backbone carbonyl oxygen of Glu85 and the amide nitrogen of Cys87. Unlike UCN-01, the basic N15 of isogranulatimide interacts with Glu17, causing a conformation change in the kinase glycine-rich loop that may contribute importantly to inhibition. The mechanism by which isogranulatimide inhibits Chk1 and its favorable kinase selectivity profile make it a promising candidate for modulating checkpoint responses in tumors for therapeutic benefit.
G2 cell cycle arrest, down-regulation of cyclin B, and induction of mitotic catastrophe by the flavoprotein inhibitor diphenyleneiodonium
Authors: Scaife Robin M.
Published: June 2022

Because proliferation of eukaryotic cells requires cell cycle–regulated chromatid separation by the mitotic spindle, it is subject to regulation by mitotic checkpoints. To determine the mechanism of the antiproliferative activity of the flavoprotein-specific inhibitor diphenyleneiodonium (DPI), I have examined its effect on the cell cycle and mitosis. Similar to paclitaxel, exposure to DPI causes an accumulation of cells with a 4N DNA content. However, unlike the paclitaxel-mediated mitotic block, DPI-treated cells are arrested in the cell cycle prior to mitosis. Although DPI-treated cells can arrest with fully separated centrosomes at opposite sides of the nucleus, these centrosomes fail to assemble mitotic spindle microtubules and they do not accumulate the Thr288 phosphorylated Aurora-A kinase marker of centrosome maturation. In contrast with paclitaxel-arrested cells, DPI impairs cyclin B1 accumulation. Release from DPI permits an accumulation of cyclin B1 and progression of the cells into mitosis. Conversely, exposure of paclitaxel-arrested mitotic cells to DPI causes a precipitous drop in cyclin B and Thr288 phosphorylated Aurora-A levels and leads to mitotic catastrophe in a range of cancerous and noncancerous cells. Hence, the antiproliferative activity of DPI reflects a novel inhibitory mechanism of cell cycle progression that can reverse spindle checkpoint-mediated cell cycle arrest.
Dual repair modulation reverses Temozolomide resistance in vitro
Authors: Barvaux Vincent A. , Ranson Malcolm , Brown Robert , McElhinney R. Stanley , McMurry T. Brian H. , Margison Geoffrey P.
Published: June 2022

Temozolomide is an alkylating agent that mediates its cytotoxic effects via O6-methylguanine (O6-meG) adducts in DNA and their recognition and processing by the postreplication mismatch repair system (MMR). O6-meG adducts can be repaired by the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (MGMT), which therefore constitutes a major resistance mechanism to the drug. Resistance to Temozolomide can also be mediated by loss of MMR, which is frequently mediated by methylation of the hMLH1 gene promoter. Methylation of hMLH1 can be reversed by treatment of cells with 5-aza-2′-deoxycytidine, while the MGMT pseudosubstrate O6-(4-bromothenyl)guanine (PaTrin-2) can deplete MGMT activity. Using a drug-resistant cell line which expresses MGMT and has methylated hMLH1, we show that while either of these treatments can individually sensitize cells to Temozolomide, the combined treatment leads to substantially greater sensitization. The increased sensitization is not observed in matched MMR proficient cells.
The dietary isothiocyanate sulforaphane targets pathways of apoptosis, cell cycle arrest, and oxidative stress in human pancreatic cancer cells and inhibits tumor growth in severe combined immunodeficient mice
Authors: Pham Nhu-An , Jacobberger James W. , Schimmer Aaron D. , Cao Pinjiang , Gronda Marcella , Hedley David W.
Published: June 2022

Anticancer effects of the dietary isothiocyanate sulforaphane were investigated in the human pancreatic cancer cell lines MIA PaCa-2 and PANC-1. Sulforaphane-treated cells accumulated in metaphase as determined by flow cytometry [4C DNA content, cyclin A(−), cyclin B1(+), and phospho-histone H3 (Ser10)(+)]. In addition, treated cells showed nuclear apoptotic morphology that coincided with an activation of caspase-8, loss of mitochondrial membrane potential, and loss of plasma membrane integrity. The initial detection of caspase-3 cleavage occurring in G2-M arrest was independent of a change in phospho-cdc2 (Tyr15) protein; consequently, sulforaphane treatment combined with UCN-01 had no significant impact on cellular toxicity. Incubations at higher sulforaphane doses (>10 μmol/L) resulted in cleavage of caspase-3 in the G1 subpopulation, suggesting that the induction of apoptosis and the sulforaphane-induced mitosis delay at the lower dose are independently regulated. Cellular toxicity in MIA PaCa-2, and to a greater extent in PANC-1, was positively correlated with a decrease in cellular glutathione levels, whereas sustained increases in glutathione observed in MIA PaCa-2 cells or the simultaneous incubation with N-acetyl-l-cysteine in PANC-1 cells were associated with resistance to sulforaphane-induced apoptosis. Daily sulforaphane i.p. injections (375 μmol/kg/d for 3 weeks) in severe combined immunodeficient mice with PANC-1 s.c. tumors resulted in a decrease of mean tumor volume by 40% compared with vehicle-treated controls. Our findings suggest that, in addition to the known effects on cancer prevention, sulforaphane may have activity in established pancreatic cancer.
Bortezomib rapidly suppresses ubiquitin thiolesterification to ubiquitin-conjugating enzymes and inhibits ubiquitination of histones and type I inositol 1,4,5-trisphosphate receptor
Authors: Xu Qun , Farah Michelle , Webster Jack M. , Wojcikiewicz Richard J.H.
Published: June 2022

The proteasome inhibitor bortezomib is an emerging anticancer agent. Although the proteasome is clearly its locus of action, the early biochemical consequences of bortezomib treatment are poorly defined. Here, we show in cultured cells that bortezomib and other proteasome inhibitors rapidly inhibit free ubiquitin levels and ubiquitin thiolesterification to ubiquitin-conjugating enzymes. Inhibition of thiolesterification correlated with a reduction in the ubiquitination of certain substrates, exemplified by a dramatic decline in histone monoubiquitination and a decrease in the rate of inositol 1,4,5-trisphosphate receptor polyubiquitination. Thus, in addition to the expected effect of blocking the degradation of polyubiquitinated substrates, bortezomib can also inhibit ubiquitination. The effect of bortezomib on histone monoubiquitination may contribute to its therapeutic actions.
Inhibitory effect of epidermal growth factor on resveratrol-induced apoptosis in prostate cancer cells is mediated by protein kinase C-α
Authors: Shih Ai , Zhang Shenli , Cao H. James , Boswell Sarah , Wu Yun-Hsuan , Tang Heng-Yuan , Lennartz Michelle R. , Davis Faith B. , Davis Paul J. , Lin Hung-Yun
Published: June 2022

Resveratrol, a naturally occurring stilbene with antitumor properties, caused mitogen-activated protein kinase [MAPK, extracellular signal-regulated kinase 1/2 (ERK1/2)] activation, nuclear translocation of Ser15-phosphorylated p53, and p53-dependent apoptosis in hormone-insensitive DU145 prostate cancer cells. Exposure of these cells to epidermal growth factor (EGF) for up to 4 hours resulted in brief activation of MAPK followed by inhibition of resveratrol-induced signal transduction, p53 phosphorylation, and apoptosis. Resveratrol stimulated c-fos and c-jun expression in DU145 cells, an effect also suppressed by EGF. An inhibitor of protein kinase C (PKC)-α, -β, and -γ (CGP41251) enhanced Ser15 phosphorylation of p53 by resveratrol in the absence of EGF and blocked EGF inhibition of the resveratrol effect. EGF caused PKC-α/β phosphorylation in DU145 cells, an effect reversed by CGP41251. Activation of PKC by phorbol ester (phorbol 12-myristate 13-acetate) enhanced EGF action on ERK1/2 phosphorylation without significantly altering p53 phosphorylation by resveratrol. DU145 cells transfected with a dominant-negative PKC-α construct showed resveratrol-induced ERK1/2 phosphorylation and Ser15 phosphorylation of p53 but were unresponsive to EGF. Thus, resveratrol and EGF activate MAPK by discrete mechanisms in DU145 cells. The stilbene promoted p53-dependent apoptosis, whereas EGF opposed induction of apoptosis by resveratrol via a PKC-α-mediated mechanism. Resveratrol also induced p53 phosphorylation in LNCaP prostate cancer cells, an effect also inhibited by EGF. Inhibition of PKC activation in LNCaP cells, however, resulted in a reduction, rather than increase, in p53 activation and apoptosis, suggesting that resveratrol-induced apoptosis in these two cell lines occurs through different PKC-mediated and MAPK-dependent pathways.
Disruption of WT1 gene expression and exon 5 splicing following cytotoxic drug treatment: Antisense down-regulation of exon 5 alters target gene expression and inhibits cell survival
Authors: Renshaw Jane , Orr Rosanne M. , Walton Michael I. , te Poele Robert , Williams Richard D. , Wancewicz Edward V. , Monia Brett P. , Workman Paul , Pritchard-Jones Kathryn
Published: June 2022

Deregulated expression of the Wilms' tumor gene (WT1) has been implicated in the maintenance of a malignant phenotype in leukemias and a wide range of solid tumors through interference with normal signaling in differentiation and apoptotic pathways. Expression of high levels of WT1 is associated with poor prognosis in leukemias and breast cancer. Using real-time (Taqman) reverse transcription-PCR and RNase protection assay, we have shown up-regulation of WT1 expression following cytotoxic treatment of cells exhibiting drug resistance, a phenomenon not seen in sensitive cells. WT1 is subject to alternative splicing involving exon 5 and three amino acids (KTS) at the end of exon 9, producing four major isoforms. Exon 5 splicing was disrupted in all cell lines studied following a cytotoxic insult probably due to increased exon 5 skipping. Disruption of exon 5 splicing may be a proapoptotic signal because specific targeting of WT1 exon 5–containing transcripts using a nuclease-resistant antisense oligonucleotide (ASO) killed HL60 leukemia cells, which were resistant to an ASO targeting all four alternatively spliced transcripts simultaneously. K562 cells were sensitive to both target-specific ASOs. Gene expression profiling following treatment with WT1 exon 5–targeted antisense showed up-regulation of the known WT1 target gene, thrombospondin 1, in HL60 cells, which correlated with cell death. In addition, novel potential WT1 target genes were identified in each cell line. These studies highlight a new layer of complexity in the regulation and function of the WT1 gene product and suggest that antisense directed to WT1 exon 5 might have therapeutic potential.
Antisense oligonucleotides targeted to the human α folate receptor inhibit breast cancer cell growth and sensitize the cells to doxorubicin treatment
Authors: Jhaveri Mona S. , Rait Antonina S. , Chung Koong-Nah , Trepel Jane B. , Chang Esther H.
Published: June 2022

Folates are essential for cell survival and are required for numerous biochemical processes. The human α isoform folate receptor (αhFR) has a very high affinity for folic acid and is considered an essential component in the cellular accumulation of folates and folate analogues used in chemotherapy. The expression of αhFR is not detected inmost normal tissues. In contrast, high levels of the expression of αhFR have been reported in a variety of cancer cells. The significance of αhFR overexpression in malignant tissues has not been elucidated, but it is possible that it promotes cell proliferation not only by mediating folate uptake but also by generating other regulatory signals. The purpose of the present study was to evaluate αhFR as a potential target for the treatment of breast cancer. Initial studies were done in nasopharyngeal carcinoma (KB) cells, which express high levels of αhFR. In KB cells, antisense oligodeoxyribonucleotides (ODN) complementary to the αhFR gene sequences were found to reduce newly synthesized αhFR protein up to 60%. To examine the effect of αhFR antisense ODNs in a panel of cultured human breast cancer cell lines, we used a tumor cell–targeted, transferrin-liposome–mediated delivery system. The data show that αhFR antisense ODNs induced a dose-dependent decrease in cell survival. Finally, we determined that αhFR antisense ODNs sensitized MDA-MB-435 breast cancer cells by 5-fold to treatment with doxorubicin. The data support the application of αhFR antisense ODNs as a potential anticancer agent in combination with doxorubicin.
Role of human copper transporter Ctr1 in the transport of platinum-based antitumor agents in cisplatin-sensitive and cisplatin-resistant cells
Authors: Song Im-Sook , Savaraj Niramol , Siddik Zahid H. , Liu Peiman , Wei Yingjie , Wu Chun Jing , Kuo Macus Tien
Published: June 2022

Recent studies have shown that the mammalian high-affinity copper transporter encoded by Ctr1 is involved in the uptake of cisplatin. However, the roles of hCtr1 in cisplatin-sensitive and cisplatin-resistant mammalian cells have not been investigated. Here, we show that, of five cisplatin-resistant cell lines, only one (SR2) exhibited substantial reduction in hCtr1 expression as compared with that in its sensitive line small cell lung cancers (SCLC), whereas copper efflux transporters ATP7A and ATP7B were not significantly altered. SR2 exhibited cross-resistance to carboplatin but not to oxaliplatin. Transfection of expression hemagglutinin-tagged hCtr1 cDNA into SCLC and SR2 cells enhanced the uptake of copper, cisplatin, carboplatin, and oxaliplatin, suggesting that hCtr1 transporter can transport these platinum-based drugs. Whereas increased sensitivities to all these platinum drugs were observed in hCtr1-transfected SCLC cells, increased sensitivities to cisplatin and carboplatin but not to oxaliplatin were observed in hCtr1-transfected SR2 cells. These results suggest that SR2 acquired an additional unique intracellular resistance mechanism to oxaliplatin. Finally, using hCtr1 deletion mutants, we showed that the NH2-terminal domain of hCtr1 was involved in transporting all these platinum-based antitumor agents. These results collectively show the importance of hCtr1 in the transport of platinum-based antitumor agents in cisplatin-sensitive and cisplatin-resistant variants.
Silymarin induces apoptosis primarily through a p53-dependent pathway involving Bcl-2/Bax, cytochrome c release, and caspase activation
Authors: Katiyar Santosh K. , Roy Anshu M. , Baliga Manjeshwar S.
Published: June 2022

Silymarin, a plant flavonoid, has been shown to inhibit skin carcinogenesis in mice. However, the mechanism responsible for the anti-skin carcinogenic effects of silymarin is not clearly understood. Here, we report that treatment of JB6 C141 cells (preneoplastic epidermal keratinocytes) and p53+/+ fibroblasts with silymarin and silibinin (a major constituent of silymarin) resulted in a dose-dependent inhibition of cell viability and induction of apoptosis in an identical manner. Silymarin-induced apoptosis was determined by fluorescence staining (8–64% apoptosis) and flow cytometry (12–76% apoptosis). The silymarin-induced apoptosis was primarily p53 dependent because apoptosis occurred to a much greater extent in the cells expressing wild-type p53 (p53+/+, 9–61%) than in p53-deficient cells (p53−/−, 6–20%). The induction of apoptosis in JB6 C141 cells was associated with increased expression of the tumor suppressor protein, p53, and its phosphorylation at Ser15. The constitutive expression of antiapoptotic proteins Bcl-2 and Bcl-xl were decreased after silymarin treatment, whereas the expression of the proapoptotic protein Bax was increased. There was a shift in Bax/Bcl-2 ratio in favor of apoptotic signal in silymarin-treated cells, which resulted in increased levels of cytochrome c release, apoptotic protease-activating factor-1, and cleaved caspase-3 and poly(ADP-ribose) polymerase in JB6 C141 cells. The shift in Bax/Bcl-2 ratio was more prominent in p53+/+ fibroblasts than in p53−/− cells. Silymarin-induced apoptosis was blocked by the caspase inhibitor (Z-VAD-FMK) in JB6 C141 cells which suggested the role of caspase activation in the induction of apoptosis. These observations show that silymarin-induced apoptosis is primarily p53 dependent and mediated through the activation of caspase-3.
Nucleotide-based therapies targeting clusterin chemosensitize human lung adenocarcinoma cells bothin vitroandin vivo
Authors: July Laura V. , Beraldi Eliana , So Alan , Fazli Ladan , Evans Kenneth , English John C. , Gleave Martin E.
Published: June 2022

Introduction: Lung cancer is highly lethal and resistant to most anticancer interventions. Treatment resistance is mediated, in part, by enhanced expression of cell survival proteins that help facilitate tumor progression. Clusterin is a stress-associated cytoprotective protein up-regulated by various apoptotic triggers in many cancers and confers treatment resistance when overexpressed. The objectives in this study were to evaluate clusterin expression levels in human lung cancer tissue, and to test effects of clusterin silencing using antisense oligonucleotides (ASOs) and short interfering double-stranded RNAs (siRNAs) on chemosensitivity in human lung cancer A549 cells. Methods: Clusterin immunostaining was evaluated in a tissue microarray of 149 spotted human lung cancers. The effects of clusterin ASO or siRNA treatment on clusterin expression and chemosensitivity to paclitaxel was examined in A549 cells in vitro while the ability of clusterin ASO to chemosensitize in vivo was evaluated in immunocompromised mice bearing A549 tumors. Results: More than 80% of human non-small cell lung cancers are immunoreactive for clusterin. Clusterin ASO or siRNA decreased clusterin mRNA expression in A549 cells >75% in a dose-dependent, sequence-specific manner, and significantly enhanced chemosensitivity to paclitaxel in vitro. Characteristic apoptotic DNA laddering was observed after combined treatment with ASO plus paclitaxel, but not with either agent alone. In vivo administration of clusterin ASO, compared to mismatch control oligonucleotide, synergistically enhanced the effects of paclitaxel or gemcitibine to significantly delay A549 tumor growth. Conclusion: These findings identify clusterin as a valid therapeutic target in strategies employing novel multimodality therapy for advanced lung cancer.
Gonadotropin-releasing hormone receptor–targeted gene therapy of gynecologic cancers
Authors: Gründker Carsten , Huschmand Nia Abdohamid , Emons Günter
Published: June 2022

The majority of ovarian, endometrial, and breast cancers express gonadotropin-releasing hormone (GnRH) receptors. Apart from reproductive organs (ovaries, fallopian tubes, and uterus) that are normally removed during surgical therapy of ovarian or endometrial cancer, pituitary gonadotrophs also express GnRH receptors. The signal transduction pathway in tumor cells is basically different from the classic GnRH receptor signal transduction, which is known to operate in the pituitary gonadotrophs and can therefore be considered tumor specific. Other organs and hematopoetic stem cells do not express GnRH receptors. We have recently shown specific activation of nucleus factor κB in ovarian, endometrial, and breast cancers after treatment with GnRH agonists. Based on this tumor-specific signaling pathway and the distribution pattern of GnRH receptors, we have developed and successfully tested a gene therapy concept by using a GnRH analogue as an inducer for the transcription of a therapeutic gene in cell culture and in nude mice.
Phosphorothioate oligodeoxynucleotides and G3139 induce apoptosis in 518A2 melanoma cells
Authors: Lai Johnathan C. , Benimetskaya Luba , Khvorova Anastasia , Wu Sijian , Hua Emily , Miller Paul , Stein C.A.
Published: June 2022

In a previous study, we showed that G3139, an antisense phosphorothioate oligonucleotide that down-regulates the expression of Bcl-2 protein, did not cause chemosensitization of 518A2 melanoma cells. In this work, we show that G3139, and the 2-base mismatch, G4126, can initiate apoptosis in this and other melanoma cell lines as shown by increased cell surface Annexin V expression, typical nuclear phenotypic changes as assessed by 4′,6-diamidino-2-phenylindole staining, activation of caspase-3 (but not caspase-8) and Bid, appearance of DEVDase (but not IETDase) activity, and cleavage of poly(ADP-ribose)-polymerase 1. Depolarization of the mitochondrial membrane occurs as a relatively late event. All of these processes seem to be substantially, but perhaps not totally, Bcl-2 independent as shown by experiments employing an anti-Bcl-2 small interfering RNA, which as shown previously down-regulated Bcl-2 protein expression but did not produce apoptosis or chemosensitization in melanoma cells. In fact, these G3139-induced molecular events were not dramatically altered in cells that forcibly overexpressed high levels of Bcl-2 protein. Addition of irreversible caspase inhibitors (e.g., the pan-caspase inhibitor zVAD-fmk) to G3139-treated cells almost completely blocked cytotoxicity. Examination of the time course of the appearance of caspase-3 and cleaved poly(ADP-ribose)-polymerase 1 showed that this could be correlated with the release of cytochrome c from the mitochondria, an event that begins only ∼4 hours after the end of the oligonucleotide/LipofectAMINE 2000 5-hour transfection period. Thus, both G3139 and cytotoxic chemotherapy activate the intrinsic pathway of apoptosis in these cells, although Bcl-2 expression does not seem to contribute strongly to chemoresistance. These findings suggest that the attainment of G3139-induced chemosensitization in these cells will be difficult.
Silatecan DB-67 is a novel DNA topoisomerase I–targeted radiation sensitizer
Authors: Chen Allan Y. , Shih Shyh-Jen , Garriques Liza N. , Rothenberg Mace L. , Hsiao Michael , Curran Dennis P.
Published: June 2022

The silatecan 7-tert-butyldimethylsilyl-10-hydroxy-camptothecin (DB-67) represents a new generation of camptothecin derivatives that exhibits a potent in vitro DNA topoisomerase I (TOP1)–mediated DNA-damaging activity, improved blood stability, and holds significant promise for the treatment of human cancers. In this study, we characterize the role of TOP1 in mediating the radiosensitization activity of DB-67. As examined by clonogenic survival assay, DB-67 exhibited potent radiosensitization activity at a concentration 10-fold lower than camptothecin in the human glioma D54-MG and T-98G cells, which harbor wild-type and mutant p53, respectively. Analyzed by the single-hit multitarget model, DB-67 induced radiosensitization by obliterating the “shoulder” of the radiation survival curve in the D54-MG cells. The in vivo targeting of TOP1 by DB-67 was investigated by immunoblot analysis. In a dose-dependent manner, DB-67 specifically stimulates covalent linking of TOP1 to chromosomal DNA at concentrations 10-fold lower than camptothecin in the D54-MG cells. The potency of in vivo targeting of TOP1 by DB-67 correlates well with its cytotoxicity and radiosensitization activity. Furthermore, DB-67 exhibited substantially less cytotoxicity and radiosensitization activity in the TOP1 mutant Chinese hamster lung fibroblast DC3F/C-10 cells than in their parental DC3F cells. Together, our data show that DB-67 exhibits potent cytotoxicity and radiosensitization activity by targeting TOP1 in mammalian cells and has great potential for being developed to treat human cancers.
Retinoic acid-induced CD38 antigen as a target for immunotoxin-mediated killing of leukemia cells
Authors: Mehta Kapil , Ocanas Larry , Malavasi Fabio , Marks John W. , Rosenblum Michael G.
Published: June 2022

A major obstacle in the successful delivery of antibody-based therapeutics to tumor cells is the heterogeneity of target antigen expression. We reported previously that retinoic acid (RA) is a potent and selective inducer of the cell-surface antigen CD38 in myeloid leukemia cells. The purpose of this study was to determine whether the RA-induced CD38 antigen could be a target for an anti-CD38-based immunotoxin to induce selective killing of leukemia cells. The combination of RA and the anti-CD38 gelonin immunotoxin induced a synergistic killing of leukemia cells. Thus, coculture of myeloid leukemia cells and cell lines with as little as 1 nm RA in the presence of immunotoxin induced substantial killing (>90%) of leukemia cell clones. More importantly, the blasts of myeloid leukemia patients, irrespective of their morphological and phenotypic features, also responded to the RA and immunotoxin combination when cultured ex vivo. A similar synergistic effect between RA and immunotoxin was observed against a multidrug-resistant variant subline of HL-60 cells. However, another variant of HL-60 cells, HL-60R, in which the retinoid receptor function has been abrogated by a trans-dominant-negative mutation, exhibited complete resistance to the immunotoxin-induced killing effect in the presence or absence of RA. Our results suggest that RA combined with anti-CD38-based therapeutic agent may offer exciting opportunities for the treatment of myeloid leukemias despite their multiplicity of genetic and clinical varieties.
The mitotic serine threonine kinase, Aurora-2, is a potential target for drug development in human pancreatic cancer
Authors: Rojanala Sangeeta , Han Haiyong , Muñoz Rubén M. , Browne Walden , Nagle Raymond , Von Hoff Daniel D. , Bearss David J.
Published: June 2022

Aurora-2 is a serine threonine kinase that associates with the centrosome. Overexpression or ectopic expression of Aurora-2 appears to alter centrosome number and function and has been implicated in a variety of human cancers. In this work, we demonstrate that Aurora-2 is both amplified and overexpressed in human pancreatic cancer cell lines, with a 2–5-fold increase in gene copy number and a 3–4-fold increase in protein levels compared with controls. Aurora-2 is also amplified and overexpressed in pancreatic cancers taken directly from patients. An immunohistochemistry of tissues taken directly from patients demonstrated an overexpression of Aurora-2 in 26 of 28 pancreatic cancers compared with 18 normal pancreas samples. Antisense nucleotides specifically targeted at Aurora-2 arrest the cell cycle in pancreatic cancer cells, indicating the potential of Aurora-2 as a therapeutic target in pancreatic cancer.
Selective inhibitors of MEK1/ERK44/42 and p38 mitogen-activated protein kinases potentiate apoptosis induction by sulindac sulfide in human colon carcinoma cells
Authors: Sun Yunjie , Sinicrope Frank A.
Published: June 2022

The nonsteroidal anti-inflammatory drug (NSAID) sulindac prevents experimental colon cancer and can regress precancerous polyps in humans. Sulindac sulfide inhibits cyclooxygenase (COX)-mediated prostaglandin synthesis and retards the growth of cultured colon cell lines primarily by inducing apoptosis. Given the known role of mitogen-activated protein kinase (MAPK) in signal transduction and the regulation of cell survival and death, we determined the effect of sulindac sulfide on MAPK activation, COX-2 expression, and apoptosis induction in HCA-7 human colon cancer cells. Sulindac sulfide treatment was associated with activation of ERKp44/42 and p38 MAPK in a dosage- and time-dependent manner, and also activated upstream MEK. Similar results were seen in HCT-15 cells and also with the selective COX-2 inhibitor NS398. ERKp44/42 and p38 activation were accompanied by an induction of COX-2 protein expression. Selective inhibitors of sulindac sulfide–induced ERKp44/42 (PD98059) and p38 MAPK (SB203580) activation also suppressed the induction of COX-2 by this NSAID. Furthermore, both MAPK inhibitors significantly augmented sulindac sulfide–induced apoptosis, as did suppression of constitutive COX-2 using antisense oligonucleotides. In conclusion, MEK/ERK and p38 MAPK activation mediate COX-2 induction by sulindac sulfide. Selective inhibitors of these MAPKs potentiate apoptosis induction by this NSAID, suggesting a novel strategy for the prevention or treatment of colorectal cancer.
The insulin-like growth factor-I (IGF-I) receptor kinase inhibitor NVP-ADW742, in combination with STI571, delineates a spectrum of dependence of small cell lung cancer on IGF-I and stem cell factor signaling
Authors: Warshamana-Greene G. Sakuntala , Litz Julie , Buchdunger Elisabeth , Hofmann Francesco , Garcı́a-Echeverrı́a Carlos , Krystal Geoffrey W.
Published: June 2022

Stem cell factor (SCF)/Kit and insulin-like growth factor-I (IGF-I)/IGF-I receptor (IGF-IR) autocrine loops play a prominent role in the growth of small cell lung cancer (SCLC). Previous data suggested that IGF-I protects cells from apoptosis induced by STI571, an efficient inhibitor of Kit signal transduction, by activating the critical phosphatidylinositol 3-kinase-Akt pathway. To determine if inhibition of IGF-IR signaling would be therapeutically relevant in SCLC, the activity of a novel kinase inhibitor of IGF-IR, NVP-ADW742 (Novartis Pharma AG, Basel, Switzerland), was characterized. Pretreatment of the H526 cell line with NVP-ADW742 inhibited IGF-IR signaling and growth with IC50 values between 0.1 and 0.4 μm. SCF-mediated Kit phosphorylation and Akt activation were inhibited with IC50 values in the 1–5 μm range. However, NVP-ADW742 affected neither hepatocyte growth factor-mediated Akt activation nor activity of constitutively active Akt. The therapeutic potential of NVP-ADW742 was assessed by determining its effect on growth of several SCLC cell lines in serum. These studies clearly delineated two populations of cell lines as determined by differential sensitivity to NVP-ADW742. One population, which lacks active SCF/Kit autocrine loops, was inhibited with IC50 values between 0.1 and 0.5 μm. A second population, which has active SCF/Kit autocrine loops, was inhibited with IC50 values in the 4–7 μm range. When these cell lines were treated with a combination of STI571 and NVP-ADW742, no advantage was seen in the former group, whereas, in the latter group, a clearly synergistic response to the combination was seen when growth, apoptosis, or Akt activation was assessed. These data demonstrate that NVP-ADW742 is a potent and selective IGF-IR kinase inhibitor that can efficiently inhibit the growth of cells that are highly dependent on IGF-I signaling. However, for optimal growth inhibition of SCLC cells with an active SCF/Kit autocrine loop, a combination of a Kit inhibitor (STI571) and an IGF-IR inhibitor (NVP-ADW742) appears to be necessary. These observations suggest that, in tumors in which critical signal transduction pathways can be activated by alternative receptors, optimal therapy may require inhibition of multiple receptors.
Simultaneous inhibition of hsp 90 and the proteasome promotes protein ubiquitination, causes endoplasmic reticulum-derived cytosolic vacuolization, and enhances antitumor activity
Authors: Mimnaugh Edward G. , Xu Wanping , Vos Michele , Yuan Xitong , Isaacs Jennifer S. , Bisht Kheem S. , Gius David , Neckers Len
Published: June 2022

The ansamycin antibiotic, geldanamycin, targets the hsp 90 protein chaperone and promotes ubiquitin-dependent proteasomal degradation of its numerous client proteins. Bortezomib is a specific and potent proteasome inhibitor. Both bortezomib and the geldanamycin analogue, 17-N-allylamino-17-demethoxy geldanamycin, are in separate clinical trials as new anticancer drugs. We hypothesized that destabilization of hsp 90 client proteins with geldanamycin, while blocking their degradation with bortezomib, would promote the accumulation of aggregated, ubiquitinated, and potentially cytotoxic proteins. Indeed, geldanamycin plus bortezomib inhibited MCF-7 tumor cell proliferation significantly more than either drug alone. Importantly, while control cells were unaffected, human papillomavirus E6 and E7 transformed fibroblasts were selectively sensitive to geldanamycin plus bortezomib. Geldanamycin alone slightly increased protein ubiquitination, but when geldanamycin was combined with bortezomib, protein ubiquitination was massively increased, beyond the amount stabilized by bortezomib alone. In geldanamycin plus bortezomib-treated cells, ubiquitinated proteins were mostly detergent insoluble, indicating that they were aggregated. Individually, both geldanamycin and bortezomib induced hsp 90, hsp 70, and GRP78 stress proteins, but the drug combination superinduced these chaperones and caused them to become detergent insoluble. Geldanamycin plus bortezomib also induced the formation of abundant, perinuclear vacuoles, which were neither lysosomes nor autophagosomes and did not contain engulfed cytosolic ubiquitin or hsp 70. Fluorescence marker experiments indicated that these vacuoles were endoplasmic reticulum derived and that their formation was prevented by cycloheximide, suggesting a role for protein synthesis in their genesis. These observations support a mechanism whereby the geldanamycin plus bortezomib combination simultaneously disrupts hsp 90 and proteasome function, promotes the accumulation of aggregated, ubiquitinated proteins, and results in enhanced antitumor activity.
Different DNA lesions trigger distinct cell death responses in HCT116 colon carcinoma cells
Authors: Bai Shaochun , Goodrich David W.
Published: June 2022

The pleiotrophic cellular response to DNA damage includes activation of cell cycle checkpoints, induction of DNA repair pathways, and initiation of programmed cell death among others. The fate of cells with damaged DNA depends on the coordination of these different responses. The clinical efficacy of genotoxic therapies is influenced by cell fate and thus by how the DNA damage response is coordinated. While a great deal has been learned about how different DNA lesions activate distinct cell cycle checkpoints and DNA repair pathways, less is known about whether the type of DNA lesion influences the qualitative and quantitative nature of the cell death response. To address this question, HCT116 colon carcinoma cells have been treated with equally cytotoxic doses of the antitumor DNA alkylating agents adozelesin or bizelesin or the DNA strand scission agent C-1027. The relative contribution of cell cycle arrest and cell death to measured cytotoxicity varied among the three drugs. Apoptotic cell death accounts for most C-1027 cytotoxicity while cell cycle arrest and cell death both contribute to the cytotoxicity of the alkylating agents. Each of the drugs induces a distinct but overlapping pattern of caspase activation. In addition, the cell death response to these drugs is differentially dependent on p53 and p21. These observations suggest that the type of DNA lesion influences not only the relative extent of apoptotic cell death at a given cytotoxic dose but also the qualitative nature of that response.
Identification of human polo-like kinase 1 as a potential therapeutic target in pancreatic cancer
Authors: Gray Phillip J. , Bearss David J. , Han Haiyong , Nagle Raymond , Tsao Ming-Sound , Dean Nicholas , Von Hoff Daniel D.
Published: June 2022

Pancreas cancer is the fourth leading cause of cancer-related death in adults in the United States. New molecular targets for diagnosis and therapy of this disease are desperately needed. In this study, we report on the mitotic serine-threonine kinase polo-like kinase 1 (Plk1) in pancreatic cancer. Plk1 mRNA was found to be overexpressed in 9 of 10 tested pancreatic cancer cell lines and in 4 of 4 tested human tumors. Immunohistochemical staining of a pancreatic tissue microarray showed that 26 of the 35 tumors taken directly from patients overexpressed Plk1. We also examined the effects of depleting Plk1 in pancreatic cancer cells by the use of antisense oligonucleotides. Antisense-treated pancreatic cancer cells showed cell cycle arrest in G2-M as well as a drastic reduction in proliferation rates. These data suggest that Plk1 is a potential therapeutic target in devising a treatment for patients with pancreatic cancer.
Interstitial gene delivery in human xenograft prostate tumors using titanium metal seeds
Authors: Jung Mira , Zhang Yin , Dimtchev Alexandre , Subramanian Manny R. , Suthanthiran Krishman , Dritschilo Anatoly
Published: June 2022

Gene therapy is a promising approach for the treatment of cancers. Strategies for gene vector delivery include systemic and local-regional approaches. Intratumoral delivery of vectors has generally employed direct injections into single or multiple locations throughout the tumor volume. However, this approach leads to nonuniform distributions of reagents within tumors and becomes cumbersome as the required number of injections is increased. We have investigated the effectiveness of an interstitial plasmid gene delivery based on using tiny metallic seeds (GeneSeeds) analogous to technology used for brachytherapy. Feasibility for interstitial use of GeneSeeds was demonstrated expressing reporter plasmids (green fluorescence protein or β-galactosidase) in human xenograft prostate tumors. Immunohistochemical analysis confirmed effective interstitial delivery, vector expression, and distributions of reporter genes within tumors. Applicability of GeneSeeds for delivery of radiosensitizing cytokines was examined by generating a cytokine [tumor necrosis factor-α (TNF-α)] expressing vector under the cytomegaloviral promoter and interstitially implanting GeneSeeds with this vector into prostate cancer tumors. TNF-α protein expression was observed around the ends of seeds and decreasing in an exponential gradient as a function of distance. The expression of TNF-α resulted in tumor growth delay of a human prostate cancer xenograft. These results demonstrate the feasibility of applying interstitial delivery of gene expressing vectors for the treatment of human cancers.