Sensitivity of human pancreatic carcinoma cell line (MIA PaCa-2) to Bisantrene and Theprubicin in vitro

Summary We tested the effect of Bisantrene (BS) and Theprubicin (THP-ADR) on cell growth of a human pancreatic carcinoma cell line (MIA PaCa-2). After 1 h exposure ID₅₀ of BS or THP-ADR was 3×10⁻⁷ and 5×10⁻⁸ M, respectively. Increasing the exposure time from 1 h to continuous exposure for 5d resulted in 11-fold decrease in ID₅₀ for BS and a 6-fold decrease for THP-ADR. Both drugs inhibited [¹⁴C]thymidine incorporation to the same extent and caused an accumulation of cells into G₂+M phase of the cell cycle. This work is supported by a gift, from Mr. Issam Fares and U.S. Public Health Service Grants AM 07114 and CA 19182.     

Expression and characterization of alkaline phosphatases during differentiation of human pancreatic cancer (Capan-1) cells in culture.

Human pancreatic cells of the Capan-1 cell line differentiate in culture. During the exponential growth phase, the cells are undifferentiated, only becoming differentiated during the stationary phase. The formation of domes in this phase is related to the exchange of water and electrolytes. The present study was designed to characterize the localization and expression of alkaline phosphatases (AP) in Capan-1 cells during growth in culture. Biochemical, cytoenzymatic and immunocytochemical methods were employed combined with light and electron microscopic examination. AP essentially of the placental type were expressed progressively during the exponential growth phase, and were seen to be distributed over the surface of the Capan-1 cells. In the stationary phase, the AP became localized on the surface of microvilli. The precipitates of the enzyme reaction highlighted regular four-bodied structures. Biochemical assays showed a progressive increase in activity of this enzyme in cells during both the exponential and stationary growth phases. However, in the stationary phase between days 7 and 8, there was a fall in enzyme activity, with a corresponding increase in this activity in the culture medium. Cytological examination indicated that this fall could be accounted for by loss of AP-positive membranes by vesiculization of apical microvilli and release of microvesicles into the culture medium. Immunoblots showed that Capan-1 cells expressed two types of AP, a placental type (70 kDa) and to a lesser extent a liver type (80 kDa). Expression of the placental type was attributed to a neoplastic derepression of the coding gene, while the liver type was assumed to be a normal gene expression of human duct cells. The placental type AP might thus serve as a marker of transformation, and the liver type as a marker of differentiation.     

Synthesis of 2-,4,-6-, and/or 7-substituted quinoline derivatives as human dihydroorotate dehydrogenase (hDHODH) inhibitors and anticancer agents: 3D QSAR-assisted design

Following our research for human dihydroorotate dehydrogenase (hDHODH) inhibitors as anticancer agents, herein we describe 3D QSAR-based design, synthesis and in vitro screening of 2-,4,-6-, and/or 7-substituted quinoline derivatives as hDHODH inhibitors and anticancer agents. We have designed 2-,4,-6-, and/or 7-substituted quinoline derivatives and predicted their hDHODH inhibitory activity based on 3D QSAR study on 45 substituted quinoline derivatives as hDHODH inhibitors, and also predicted toxicity. Designed compounds were docked into the binding site of hDHODH. Designed compounds which showed good predictive activity, no toxicity, and good docking score were selected for the synthesis, and in vitro screening as hDHODH inhibitors in an enzyme inhibition assay, and anticancer agents in MTT assay against cancer cell lines (HT-29 and MDA-MB-231). Synthesized compounds 7 and 14 demonstrated IC₅₀ value of 1.56?µM and 1.22?µM, against hDHODH, respectively, and these are our lead compounds for the development of new hDHODH inhibitors and anticancer agents.     

The establishment of human anti-TROP2 antibody IgG and its inhibition function on pancreatic cancer cell proliferation

On the basis of anti-TROP2 Fab antibody, this study seek to construct a eukaryotic expression system of human anti-TROP2 antibody IgG, and to analyse the inhibition function of human anti-TROP2 antibody IgG in the cell proliferation of pancreatic cancer. The heavy and light chain genes of anti-TROP2 antibody were amplified respectively to establish the recombinant expression vector of human anti-TROP2 antibody IgG, named pWS-anti-TROP2. The human anti-TROP2 antibody IgG was obtained through transfecting the plasmids into the CHO dhfr^- cell line, selecting the monoclonal cell strains with high amounts of antibody expression by MTX screening and applying Protein G affinity in purification. The identification and immunologic activity of human anti-TROP2 antibody IgG were researched by Western Blot,SDS-PAGE, ELISA, immunofluorescence assay and flow cytometry method (FCM). MTT assay was conducted to analyse the inhibition effect of human anti-TROP2 antibody IgG on BxPC3 cell proliferation. The human anti-TROP2 antibody IgG eukaryotic expression system was established successfully to express human anti-TROP2 antibody IgG, in which the molecular weight of heavy chain and light chain were consistent with expectation, and it could specifically combine with TROP2 protein, the antibody titer reached 1:6,400. The MTT assay results indicated that human anti-TROP2 antibody IgG had a significant effect on inhibiting the proliferation of BxPC3 cell, and the inhibition function can be gradually increased with improved antibody dose and prolonged time. In the study, the human anti-TROP2 antibody IgG eukaryotic expression system was constructed successfully, the antibody could specifically bind to TROP2 protein on the surface of pancreatic cancer cells, and it is shown to have a significant inhibitory action in pancreatic cancer cell proliferation.     

Evaluation of the potential therapeutic role of a new generation of vitamin D analog,MART-10, in human pancreatic cancer cells in vitro and in vivo

Pancreatic cancer is a lethal disease with no known effective chemotherapy and radiotherapy, and most patients are diagnosed in the late stage, making them unsuitable for surgery. Therefore, new therapeutic strategies are urgently needed. 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] is known to possess antitumor actions in many cancer cells in vitro and in vivo models. However, its clinical use is hampered by hypercalcemia. In this study, we investigated the effectiveness and safety of a new generation, less calcemic analog of 1α,25(OH)₂D₃, 19-nor-2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D₃ (MART-10), in BxPC-3 human pancreatic carcinoma cells in vitro and in vivo. We demonstrate that MART-10 is at least 100-fold more potent than 1α,25(OH)₂D₃ in inhibiting BxPC-3 cell proliferation in a time- and dose-dependent manner, accompanied by a greater upregulation of cyclin-dependent kinase inhibitors p21 and p27 and a greater downregulation of cyclin D3 and cyclin-dependent kinases 4 and 5, leading to a greater increase in the fraction of cells in G₀/G₁ phase. No induction of apoptosis and no effect on Cdc25 phosphatases A and C were observed in the presence of either MART-10 or 1α,25(OH)₂D₃. In a xenograft mouse model, treatment with 0.3 µg/kg body weight of MART-10 twice/week for 3 weeks caused a greater suppression of BxPC-3 tumor growth than the same dose of 1α,25(OH)₂D₃ without inducing hypercalcemia and weight loss. In conclusion, MART-10 is a promising agent against pancreatic cancer growth. Further clinical trial is warranted.     

Modification of blood group A antigen expression in a pancreatic cancer cell line (PC-1) by inhibitors of N-glycan processing.

Pancreatic adenocarcinomas induced in Syrian hamsters by treatment with N-nitrosobis(2-oxopropyl) amine express blood group A antigen, which is absent in normal pancreatic cells. On membrane glycoproteins purified from tumors, blood group A antigen has been found to be expressed on multiantennary Asn-linked complex glycans. In this study, we investigated the effect of inhibitors of Asn-glycan processing on blood group A antigen bearing glycan structures in a cell line (PC-1) established from a primary induced pancreatic cancer. Expression of blood group A antigen on cells and in membrane preparations was blocked by treatment with 1-deoxymannojirimycin, an inhibitor of mannosidase I, but was retained after treatment with swainsonine, an inhibitor of mannosidase II. However, swainsonine treatment altered the glycan structure associated with blood group A antigen from an endoglycosidase H resistant type to a sensitive type, indicating that the blood group A structure might shift from a complex type to a hybrid type glycan by this treatment. These results demonstrate that Asn-linked glycans carry the major blood group A antigens in PC-1 cells.     

Synthesis and structure–activity relationships of N-aryl(indol-3-yl)glyoxamides as antitumor agents

The synthesis and study of the structure–activity relationships of cytotoxic compounds based on N-pyridinyl or N-aryl-2-(1-benzylindol-3-yl)glyoxamide skeleton, represented by the lead structures D-24241 and D-24851, are described. The presence of N-(pyridin-4-yl) moiety was crucial for activity and 2-[1-(4-chloro-3-nitrobenzyl)-1H-indol-3-yl]-2-oxo-N-(pyridin-4-yl)acetamide (55), the most potent derivative, showed IC₅₀ = 39 nM, 51 nM and 11 nM against HeLa/KB (human cervix carcinoma), L1210 (murine leukemia) and SKOV3 (human ovarian carcinoma) cell lines proliferation assay, respectively, as active as the lead compounds.     

The expression of carbonic anhydrases II and IV in the human pancreatic cancer cell line (Capan 1) is associated with bicarbonate ion channels

Summary— Human pancreatic ductal cells of the Capan 1 cell line differentiate progressively during growth. After the exponential growth phase, the cells elongate and become polarized with their apical poles covered by microvilli and separated from the basolateral pole by tight junctions. In this stationary phase, they form domes, which are thought to result from the exchange of water and electrolytes. In this study, we demonstrated, using patch-clamp techniques, that HCO₃^? ions exit via the g350 high conductance anionic channel we observed recently at the Capan 1 cell surface. This g350 channel was thought to be either a Cl^?/HCO₃^? antiport or a simple HCO₃^? channel. The stilbene derivatives 4-acetamido-4 isothiocyano-2-2′-disulfonic acid (SITS) and 4,4′ diisothiocyano stilbene-2,2′ disulfonic acid (DIDS) reduced both the number of domes and the Cl^? and HCO₃^? flux through the g350 channel. Moreover, using histochemical, immunocytochemical and biochemical methods we showed that Capan 1 cells express a specific pattern of carbonic anhydrases (CA). Two types of CA were detected: the CA II isozyme mainly localized in the cytoplasm, but also found beneath the inner leaflet of the apical plasma membrane, and the CA IV isozyme localized on the outer leaflet of the apical plasma membrane and microvilli. Their molecular masses were 30 (CA II) and 55 kDa (CA IV), respectively. They were expressed continuously during the exponential growth phase, although their activity increased greatly during the stationary phase. Inhibition of dome formation by acetazolamide indicated the existence of a direct relationship between dome formation and CA. Characteristic structures with a central electron-dense core surrounded by a light halo were observed on the surface of cell membranes using histochemical and immunocytochemical methods. These structures were thought to represent a channel, corresponding possibly to CA IV. Our observations suggest that Capan 1 cells, despite their neoplasic transformation, produce HCO₃^? ions in the same way as normal human pancreatic ductal cells. Capan 1 cells in culture may therefore represent a suitable model for studying pancreatic duct HCO₃^? secretion at the cellular and molecular levels.     

Production of bioactive recombinant human Eb-peptide of pro-IGF-I and identification of binding components from the plasma membrane of human breast cancer cells (MDA-MB-231)

E-peptide of the pro-Insulin-like growth factor-I (pro-IGF-I) is produced from pre-pro-IGF-I by proteolytic cleavage in the post-translational processing. The human Eb-peptide (hEb-peptide), derived from the E domain of pro-IGF-IB isoform, is a bioactive molecule whose exact physiological role remains elusive. Accumulated evidence reported from our laboratory indicated that hEb-peptide possesses activity against multiple hallmark characteristics of solid tumor in different cancer cell types. In human breast carcinoma cells (MDA-MB-231), it was demonstrated that hEb-peptide can promote cell attachment to substratum, inhibit colony formation in a semisolid medium, reduce cancer cell invasion, and inhibit cancer-induced angiogenesis. Like the action of other peptide hormones, these cellular responses triggered by hEb may be initiated through binding to a receptor molecule residing on the surface of the cell. Our laboratory and the others have previously provided evidence demonstrating the existence of hEb-peptide specific binding components residing on the cell membrane. In this study, we report the isolation and identification of eight protein molecules bound reversibly with hEb-peptide from the membrane preparation of MDA-MB-231 cells. Some of the identified proteins are known to be present at cell surface and function as receptors while the others are not. The functions of these molecules reveal strong correlation with the demonstrated activities of hEb-peptide on MDA-MB-231cells, suggesting hEb-peptide activity on cancer cells might be mediated by these molecules.     

Synthesis of 17beta-estradiol-linked platinum(II) complexes and their cytocidal activity on estrogen-dependent and -independent breast tumor cells

The synthesis of two new highly potent 17beta-estradiol-linked platinum(II) complexes is described. The new molecules are linked at position 16 of the steroid nucleus with an alkyl chain. They are made from estrone in nine chemical steps with an overall yield exceeding 10%. The biological activity of these compounds was evaluated in vitro on estrogen dependent and independent (ER(+) and ER(-)) human breast tumor cell lines: MCF-7 and MDA-MB-231. The novel compounds prove to be highly cytotoxic against breast cancer cell lines. The most cytotoxic derivative shows high affinity for the estrogen receptor alpha.     

Establishment and characterization of two new human ovarian cancer cell lines UWOV1 and UWOV2 and a subline UWOV2 (Sf) growing in serum-free conditions: Growth characteristics,biochemical, and cytogenetic studies

Summary The establishment, growth, and characterization of two new continuously growing human ovarian cancer cell lines (UWOV1 and UWOV2) as, well as a subline (UWOV2, Sf) grown in chemically defined, serum-free medium are described. The cell lines were derived from ascitic tumors of two patients suffering from cystadenocarcinomas of the ovary. Both UWOV1 and UWOV2 lines grow in anchorage-dependent fashion as monolayers, whereas UWOV2 (Sf) forms multilayered domelike structures. Cytogenetic studies revealed nonrandom abnormalities involving chromosomes 1 and 11 in all three cell lines. Secretion of soluble collagen was detected in all three cell lines. In addition, UWOV2 (Sf) produces and secretes large amounts of extracellular matrix material with an ordered fibrillar structure which may function as an attachment factor for the serum-free cells. These cell lines seem to be useful for further studies of the biology of human ovarian cancer. This research was supported by grants from National Cancer Association (S. A.) and Bekker Trust Foundation.     

Influence on antiproliferative activity of structural modification and conjugation of gonadotropin-releasing hormone (GnRH) analogues

The effect of various GnRH analogues, and their conjugates on proliferation, clonogenicity and cell cycle phase distribution of MCF-7 and Ishikawa human cancer cell lines was studied. GnRH-III, a sea lamprey GnRH analogue reduced cell proliferation by 35% and clonogenicity by 55%. Structural modifications either decreased, or did not alter biological activity. Conjugation of GnRH analogues including MI-1544, MI-1892, and GnRH-III with poly(N-vinylpyrrolidone-co-maleic acid) (P) through a tetrapeptide spacer GFLG(X) substantially increased the inhibitory effect of the GnRH analogues. The conjugate P-X-GnRH-III induced significant accumulation of cells in the G2/M phase; from 8% to 15.6% at 24 h and 9.8% to 15% at 48 h. It was concluded that conjugation of various GnRH analogues substantially enhanced their antiproliferative activity, strongly reduced cell clonogenicity and retarded cell progression through the cell division cycle at the G2/M phase.     

Comparison of efficacy of Salmonella typhimurium A1-R and chemotherapy on stem-like and non-stem human pancreatic cancer cells

The XPA1 human pancreatic cancer cell line is dimorphic, with spindle stem-like cells and round non-stem cells. We report here the in vitro IC₅₀ values of stem-like and non-stem XPA1 human pancreatic cells cells for: (1) 5-fluorouracil (5-FU), (2) cisplatinum (CDDP), (3) gemcitabine (GEM), and (4) tumor-targeting Salmonella typhimurium A1-R (A1-R). IC₅₀ values of stem-like XPA1 cells were significantly higher than those of non-stem XPA1 cells for 5-FU (P = 0.007) and CDDP (P = 0.012). In contrast, there was no difference between the efficacy of A1-R on stem-like and non-stem XPA1 cells. In vivo, 5-FU and A1-R significantly reduced the tumor weight of non-stem XPA1 cells (5-FU; P = 0.028; A1-R; P = 0.011). In contrast, only A1-R significantly reduced tumor weight of stem-like XPA1 cells (P = 0.012). The combination A1-R with 5-FU improved the antitumor efficacy compared with 5-FU monotherapy on the stem-like cells (P = 0.004). The results of the present report indicate A1-R is a promising therapy for chemo-resistant pancreatic cancer stem-like cells.     

Stereoselective synthesis of some methyl-substituted steroid hormones and their in vitro cytotoxic activity against human gastric cancer cell line MGC-803

A series of 3-, 7-, 15-, and 16-methyl-substituted steroid analogs were synthesized via a highly stereoselective 1,6-conjugate addition. Under the catalysis of CuBr, AlMe₃ reacted with four steroid dienone precursors to afford either the corresponding α-epimer of C-3 and C-7 methyl-substituted steroids as the major products, and the ratio of α/β was up to 10/1. No β-epimer has been detected for methyl addition at C-16. However, under the same reaction conditions, enantioselective methyl addition at C-15 afforded the 15β-epimer as the major product. The preliminary SAR analysis showed that the methyl substituents at C-7α and C-15β positions lead to a dramatical increase in potency against human gastric cancer cell line MGC-803.     

Effect of hyperthermia and chemotherapeutic agents on TRAIL-induced cell death in human colon cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In the previous study [Yoo and Lee, 2007], we have reported that hyperthermia could enhance the cytotoxicity of TRAIL-induced apoptosis. We observed in human colorectal cancer cell line CX-1 that TRAIL-induced apoptotic death and also that mild hyperthermia promoted TRAIL-induced apoptotic death through caspase activation and cytochrome-c release. Although its effects in vivo are not clear, hyperthermia has been used as an adjunctive therapy for cancer. Hyperthermia is often accompanied by chemotherapy to enhance its effect. In this study, CX-1 colorectal adenocarcinoma cells were treated with TRAIL concurrently with hyperthermia and oxaliplatin or melphalan. To evaluate the cell death effects on tumor cells via hyperthermia and TRAIL and chemotherapeutic agents, FACS analysis, DNA fragmentation, and immunoblottings for PARP-1 and several caspases and antiapoptotic proteins were performed. Activities of casapse-8, caspase-9, and caspase-3 were also measured in hyperthermic condition. Interestingly, when analyzed with Western blot, we detected little change in the intracellular levels of proteins related to apoptosis. Clonogenic assay shows, however, that chemotherapeutic agents will trigger cancer cell death, either apoptotic or non-apoptotic, more efficiently. We demonstrate here that CX-1 cells exposed to 42 degrees C and chemotherapeutic agents were sensitized and died by apoptotic and non-apoptotic cell death even in low concentration (10 ng/ml) of TRAIL.     

Cx31.1 acts as a tumour suppressor in non-small cell lung cancer (NSCLC) cell lines through inhibition of cell proliferation and metastasis

Reduced connexin expression and loss of gap junction function is a characteristic of many cancers, including lung cancer. However, there are little reports about the relation between Cx31.1 and lung cancer. This study was conducted to investigate the effect of Cx31.1 on non-small cell lung cancer (NSCLC). We found that the Cx31.1 was down-regulated in NSCLC cell lines, and the expression levels were reversely related with their metastatic potential. We ectopically expressed Cx31.1 in H1299 NSCLC cell line to examine the influence of Cx31.1 overexpression. The results showed that overexpression of Cx31.1 in H1299 cells reduced cell proliferation, induced a delay in the G(1) phase, inhibited anchorage-independent growth and suppressed cell migration and invasion. The cell cycle delay and cell migration and invasion suppressive effects of Cx31.1 were partially reversed by siRNA targeting mRNA of Cx31.1. Moreover, xenografts of Cx31.1 overexpressing H1299 cells showed reduced tumourigenicity. These results suggested that Cx31.1 has tumour-suppressive properties. Further investigation indicated that cyclin D3 may be responsible for Cx31.1-induced G(1) phase delay. Importantly, Cx31.1 increased the expression of epithelial markers, such as cytokeratin 18, and decreased expression of mesenchymal markers, such as vimentin, indicating a Cx31.1-mediated partial shift from a mesenchymal towards an epithelial phenotype. We concluded that Cx31.1 inhibit the malignant properties of NSCLC cell lines, the mechanisms under this may include regulation of EMT.     

The effect of a hyposmotic shock and purinergic agonists on K(Rb) efflux from cultured human breast cancer cells

The effect of a hyposmotic shock and extracellular ATP on the efflux of K⁺(Rb⁺) from human breast cancer cell lines (MDA-MB-231 and MCF-7) has been examined. A hyposmotic shock increased the fractional efflux of K⁺(Rb⁺) from MDA-MB-231 cells via a pathway which was unaffected by Cl⁻ replacement. Apamin, charybdotoxin or removing extracellular Ca²⁺ had no effect on volume-activated K⁺(Rb⁺) efflux MDA-MB-231 cells. An osmotic shock also stimulated K⁺(Rb⁺) efflux from MCF-7 cells but to a much lesser extent than found with MDA-MB-231 cells. ATP-stimulated K⁺(Rb⁺) efflux from MDA-MB-231 cells in a dose-dependent fashion but had little effect on K⁺(Rb⁺) release from MCF-7 cells. ATP-stimulated K⁺(Rb⁺) efflux was only inhibited slightly by replacing Cl⁻ with NO₃⁻. Removal of external Ca²⁺ during treatment with ATP reduced the fractional efflux of K⁺(Rb⁺) in a manner suggesting a role for cellular Ca²⁺ stores. Charybdotoxin, but neither apamin nor iberiotoxin, inhibited ATP-stimulated K⁺(Rb⁺) release from MDA-MB-231 cells. Suramin inhibited the ATP-activated efflux of K⁺(Rb⁺). UTP also stimulated K⁺(Rb⁺) efflux from MDA-MB-231 cells whereas ADP, AMP and adenosine were without effect. A combination of an osmotic shock and ATP increased the fractional efflux of K⁺(Rb⁺) to a level greater than the sum of the individual treatments. It appears that the hyposmotically-activated and ATP-stimulated K⁺ efflux pathways are separate entities. However, there may be a degree of ‘crosstalk’ between the two pathways.     

Novel lawsone-containing ruthenium(II) complexes: Synthesis,characterization and anticancer activity on 2D and 3D spheroid models of prostate cancer cells

This study describes a series of newly synthesized phosphine/diimine ruthenium complexes containing the lawsone as bioligand with enhanced cytotoxicity against different cancer cells, and apoptosis induction in prostatic cancer cells DU-145. The complexes [Ru(law)(N-N)₂]PF₆ where N-N is 2,2′-bipyridine (1) or 1,10-phenanthroline (2) and [Ru(law)(dppm)(N-N)]PF₆, where dppm means bis(diphenylphosphino)methane, N-N is 2,2′-bipyridine (3) or 1,10-phenanthroline (4), and law is lawsone, were synthesized and fully characterized by elemental analysis, molar conductivity, NMR, UV–vis, IR spectroscopies and cyclic voltammetry. The interaction of the complexes (1–4) with DNA was evaluated by circular dichroism, gel electrophoresis, and fluorescence, and the complexes presented interactions by the minor grooves DNA. The phosphinic series of complexes exhibited a remarkably broad spectrum of anticancer activity with approximately 34-fold higher than cisplatin and 5-fold higher than doxorubicin, inhibiting the growth of 3D tumor spheroids and the ability to retain the colony survival of DU-145 cells. Also, the complex (4) inhibits DU-145 cell adhesion and migration potential indicating antimetastatic properties. The mechanism of its anticancer activity was found to be related to increased reactive oxygen species (ROS) generation, increased the BAX/BCL-2 ratio and subsequent apoptosis induction. Overall, these findings suggested that the complex (4) could be a promising candidate for further evaluation as a chemotherapeutic agent in the prostate cancer treatment.