scholarly journals LDH-A Knockdown: Changes in The LDH Isoenzyme Profile and Variability in Glioma Response

2021 ◽  
Author(s):  
Masahiro Shindo ◽  
Masatomo Maeda ◽  
Ko Myat ◽  
Mayuresh Mane ◽  
Ivan J. Cohen ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Cell Lines ◽  
Nude Mice ◽  
Doubling Time ◽  
Growth Delay ◽  
Ldh Isoenzyme ◽  
Segmentation Analysis ◽  
Major Energy Source ◽  
Lactate Formation

Abstract Background: Lactate metabolism in tumors is now recognized as a major energy source and a major gluconeogenic precursor for many tumors, as well as shown to exhibit signaling properties. There is less information on the role of the LDH/lactate axis in brain tumors, although lactate formation in gliomas is associated with poor survival. Methods: Three murine glioma cell lines (GL261, CT2A, and ALTS1C1) were transduced to knockdown (KD) expression of the murine LDH-A gene. The effects of the LDH-A KD were compared to those in control (NC) cells and tumors. Results: Differences in the expression of LDH-A and LDH-B mRNA, protein, and enzymatic activity were observed in the six cell lines. LDH zymography showed a major difference in LDH subunit distribution between GL261 LDH-A KD and NC tumors, whereas little or no effect of LDH-A KD was observed in CT2A and ALTS1C1 tumors. Tumors LDH-A and LDH-B immunohistochemistry and a Weka segmentation analysis were consistent with isoenzyme patterns and the above analyses. An “inverse” LDH-A/LDH-B staining relationship (high vs low) was observed in many local GL261 tumor regions. In contrast, CT2A tumors showed a more “direct” local LDH-A/LDH-B staining relationship. LDH-A KD prolonged the doubling time of GL261 cells in culture and prevented the formation of subcutaneous flank tumors in immune-competent C57BL/6 mice (GL261 NC tumors had a prolonged growth delay). In nude mice, both LDH-A KD and NC GL261 tumors grew more rapidly than GL261 NC tumors in C57BL/6 mice. No differences between NC and KD cell proliferation (in vitro) and tumor growth in C57BL/6 mice (doubling time) were observed for CT2A and ALTS1C1 cells and tumors, consistent with the absence of a difference in their LDH isoenzyme profiles. Conclusions: These results show the combined impact of a genetic alteration (LDH-A depletion) on the LDH isoenzyme profile, expression of LDH-A vs LDH-B and LDH enzymatic activity, and the immune system (C57BL/6 vs nude mice) on the growth of s.c. located tumors.

Satraplatin, an oral platinum analog, is active and synergistic with paclitaxel and docetaxel in prostate carcinoma models

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 14620-14620 ◽  
Author(s):  
L. Lamphere ◽  
F. Obermayr ◽  
M. Caligiuri ◽  
G. Unteregger ◽  
M. S. Rudoltz ◽  
...  
Keyword(s):  
Cell Lines ◽  
Nude Mice ◽  
Prostate Carcinoma ◽  
Phase Iii ◽  
Human Prostate ◽  
Growth Delay ◽  
Phase Ii Trials ◽  
Pivotal Phase

14620 Background: Satraplatin is a novel oral platinum analog with potent cytotoxic and antitumor activity in preclinical models. Satraplatin showed activity in hormone refractory prostate cancer (HRPC) and other tumor types in Phase II trials. A pivotal Phase III trial evaluating satraplatin as 2nd-line therapy for HRPC completed accrual of > 900 patients in 2005. Satraplatin’s activity, safety profile and ease of administration make it attractive for combination regimens. Methods: Satraplatin and its active metabolite JM-118 were tested in vitro as single agents in the androgen-sensitive LNCaP and the androgen-insensitive PC-3 and DU-145 human prostate carcinoma (ca.) cell lines. For in vitro combination studies, PC-3 cells were treated with satraplatin or JM-118 either prior to, after, or concomitantly with paclitaxel or docetaxel. The PC-3 cell line was used for in vivo xenograft experiments in nude mice. Paclitaxel was given intravenously on Day 1, satraplatin orally on Days 2 to 6, and paclitaxel again on Day 8. Results: Satraplatin and JM-118 as single agents inhibited the growth of all three prostate ca. cell lines in vitro in a dose dependent fashion. IC50 values for JM-118 were < 1μM. Strong synergism was noted when PC-3 tumor cells were treated in vitro with paclitaxel or docetaxel followed by satraplatin or JM-118. Satraplatin administered orally inhibited the growth of PC-3 xenografts in nude mice. Treatment of advanced PC-3 tumors with paclitaxel (40 mg/kg) and satraplatin (35 mg/kg) was well tolerated and resulted in a Tumor Growth Delay equivalent to 3 Log Cell Kill, an effect superior to that of the single agents. Conclusions: In vitro, satraplatin and its metabolite JM-118 are active as single agents against human prostate ca. cells, and are synergistic with taxanes. In vivo, treatment with paclitaxel followed by satraplatin showed synergism without increased toxicity. These preclinical data support ongoing Phase I and II clinical trials that are evaluating combinations of satraplatin with paclitaxel or docetaxel. [Table: see text]

scholarly journals Identification of Hsp90 inhibitors as potential drugs for the treatment of TSC1/TSC2 deficient cancer

2021 ◽  
Author(s):  
Evelyn M. Mrozek ◽  
Vineeta Bajaj ◽  
Yanan Guo ◽  
Izabela Malinowska ◽  
Jianming Zhang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Xenograft Model ◽  
Growth Delay ◽  
Hsp90 Inhibitors ◽  
Null Cell ◽  
Standard Dosage

Inactivating mutations in either TSC1 or TSC2 cause Tuberous Sclerosis Complex, an autosomal dominant disorder, characterized by multi-system tumor and hamartoma development. Mutation and loss of function of TSC1 and/or TSC2 also occur in a variety of sporadic cancers, and rapamycin and related drugs show highly variable treatment benefit in patients with such cancers. The TSC1 and TSC2 proteins function in a complex that inhibits mTORC1, a key regulator of cell growth, which acts to enhance anabolic biosynthetic pathways. In this study, we identified and validated five cancer cell lines with TSC1 or TSC2 mutations and performed a kinase inhibitor drug screen with 197 compounds. The five cell lines were sensitive to several mTOR inhibitors, and cell cycle kinase and HSP90 kinase inhibitors. The IC50 for Torin1 and INK128, both mTOR kinase inhibitors, was significantly increased in three TSC2 null cell lines in which TSC2 expression was restored.  Rapamycin was significantly more effective than either INK128 or ganetespib (an HSP90 inhibitor) in reducing the growth of TSC2 null SNU-398 cells in a xenograft model. Combination ganetespib-rapamycin showed no significant enhancement of growth suppression over rapamycin. Hence, although HSP90 inhibitors show strong inhibition of TSC1/TSC2 null cell line growth in vitro, ganetespib showed little benefit at standard dosage in vivo. In contrast, rapamycin which showed very modest growth inhibition in vitro was the best agent for in vivo treatment, but did not cause tumor regression, only growth delay.

Glutathione S-transferases in tracheobronchial epithelium

1995 ◽  
Vol 269 (4) ◽  
pp. L473-L481
Author(s):  
P. M. Reddy ◽  
C. P. Tu ◽  
R. Wu
Keyword(s):  
Cell Differentiation ◽  
Environmental Factors ◽  
Enzymatic Activity ◽  
Cell Lines ◽  
Mucous Cell ◽  
Collagen Gel ◽  
Bronchial Epithelium ◽  
High Level

The purpose of this study is to characterize glutathione S-transferase (GST) gene expression in airway epithelium both in vivo and in vitro. Immunohistochemical staining of nonhuman primate lungs of well-controlled healthy animals reveals the presence of alpha- and pi-class GST isoenzymes in ciliated bronchial epithelium. The stain of mu-GST antibody is either very low or absent in some of these monkey lungs. We observed that primary tracheobronchial epithelial (TBE) cells isolated from human and monkey pulmonary tissues maintain a relatively high level of GST enzymatic activity in culture, compared with various immortalized human TBE cell lines and other nonpulmonary cell lines. Northern blot analysis demonstrated the presence of mu-, pi-, and microsomal-GST messages but not the alpha-class message in cultures of primary TBE cells as well as in various human TBE cell lines. The expression of mu- and pi-class GST genes can be further regulated in culture by various environmental factors; however, most of these regulating factors are associated with TBE cell differentiation in culture. For instance, vitamin A treatment, which was shown to enhance mucous cell differentiation in vitro, stimulated the message levels of mu- and pi-class GST. Furthermore, plating cells on collagen gel substrata, which also enhanced mucous cell differentiation in culture, instead of plastic culture surface, enhanced total GST enzymatic activity by eightfold, and this enhancement is related to an increase in the expression of the pi-class GST gene. These results demonstrated that GST genes are differentially expressed and regulated by various environmental factors in primary TBE cells and various cell lines, and the regulation is correlated to the mucous cell differentiation in culture.

CNS-9, a Novel Specific Inhibitor of ABL Tyrosine Kinase Overcomes Resistance Mechanism of Imatinib.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 761-761 ◽  
Author(s):  
Shinya Kimura ◽  
Hidekazu Segawa ◽  
Junya Kuroda ◽  
Takeshi Yuasa ◽  
Taira Maekawa
Keyword(s):  
Tyrosine Kinase ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Nude Mice ◽  
Philadelphia Chromosome ◽  
Specific Inhibitor ◽  
Vitro Assay ◽  
Abl Tyrosine Kinase

Abstract Imatinib mesylate (also known as STI-571 and Gleevec) has drastically changed the treatment of Philadelphia chromosome positive (Ph+) leukemias. However, the resistance to imatinib has frequently been reported, particularly in patients with advanced-stage disease. A novel orally bioavailable inhibitor of the ABL tyrosine kinase (TK) named CNS-9 was developed from the 2-(phenylamino)pyrimidine class to overcome resistance mechanisms of imatinib. Inhibition of TK phosphorylation (IC50) on wild type (wt) BCR/ABL in 293T cell line by CNS-9 was 22nM, which was 2-log more potent than imatinib. Importantly, CNS-9 inhibited TK phosphorylation of E255K mutant BCR/ABL with IC50 of 98nM, while imatinib could not inhibit it with clinically relevant concentration. The T315I mutant BCR/ABL protein was resistant to CNS-9 and imatinib. CNS-9 also inhibited TK phosphorylation of platelet-derived growth factor receptor (PDGFR) or c-Kit pathways at the very similar observed IC50s when compared with imatinib, in spite of significant higher potency against ABL. The ability of CNS-9 in vitro to inhibit 101 TK molecules was assayed by KinaseProfilerTM (Upstate), showing also more specific inhibitory activity against ABL than imatinib. The growth of BCR/ABL-positive cell lines K562, KU812, BaF3 harboring wt BCR/ABL (BaF3/wt) and E255K (BaF3/E255K) was inhibited by CNS-9 with IC50 of 5, 3, 17, and 110nM, respectively (Table 1). Generally, CNS-9 was 20 to 30-fold more potent on the growth inhibition than imatinib in these same cell lines. We next investigated the in vivo effect on the leukemic growth inhibition of CNS-9. Nude mice were injected subcutaneously with 3x107 KU812 (wt BCR/ABL) on Day 0. CNS-9 or imatinib were orally administrated twice a day from Day 7 to Day 18. The dosages of CNS-9 and imatinib, which inhibited completely tumor growth were 20mg/kg/day and 200mg/kg/day, respectively, indicating that CNS-9 is 10-fold potent than imatinib in vivo. To examine the in vivo effect of CNS-9 against mutant BCR/ABL, BaF3/wt, BaF3/E255K or BaF3/T315I were engrafted to nude mice and treated with CNS-9 or imatinib. CNS-9 was also 10-fold potent than imatinib against BaF3/wt. Intriguingly, mice harboring BaF3/wt or BaF3/E255K showed significantly prolonged survival when treated with CNS-9. Consistent with in vitro assay, CNS-9 had no effect on T315I, and imatinib was not effective against both E255K and T315I. In conclusion, CNS-9 is substantially more inhibitory and more specifically than imatinib toward BCR/ABL-dependent cell growth both in vitro and in vivo Moreover, CNS-9 may be effective for leukemia patients whose leukemic cells harbor E255K mutant. The efficacy and safety of CNS-9 for Ph+ leukemias should be verified in early phase clinical trials. The IC50s values of leukemic cell lines for CNS-9 and imatinib CNS-9 (nM) imatinib (nM) K562 p210 wt BCR/ABL 5 130 KU812 p210 wt BCR/ABL 3 67 U937 BCR/ABL (−) >1000 >1000 BaF3 p190 wt BCR/ABL 17 360 BaF3 p190 E255K BCR/ABL 110 >1000 BaF3 p190 T315I BCR/ABL >1000 >1000

The Monoclonal Antibody nBT062 Conjugated to Cytotoxic Maytansinoids Has Potent and Selective Cytotoxicity against CD138 Positive Multiple Myeloma Cells in Vitro and in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1716-1716 ◽  
Author(s):  
Hiroshi Ikeda ◽  
Teru Hideshima ◽  
Robert J. Lutz ◽  
Sonia Vallet ◽  
Samantha Pozzi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Tumor Cells ◽  
Cell Lines ◽  
Xenograft Model ◽  
Growth Delay ◽  
Selective Cytotoxicity ◽  
Bystander Killing

Abstract CD138 is expressed on differentiated plasma cells and is involved in the development and/or proliferation of multiple myeloma (MM), for which it is a primary diagnostic marker. In this study, we report that immunoconjugates comprised of the murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives (nBT062-SMCC-DM1, nBT062-SPDB-DM4 and nBT062-SPP-DM1) showed cytotoxic activity against CD138-positive MM cells both in vitro and in vivo. These agents demonstrated cytotoxicity against OPM1 and RPMI8226 (CD138-positive MM cell lines) in a dose and time-dependent fashion and were also cytotoxic against primary tumor cells from MM patients. Minimal cytotoxicity was noted in CD138-negative cell lines and no activity was observed against peripheral blood mononuclear cells from healthy volunteers, suggesting that CD138-targeting is important for immunoconjugate-mediated cytotoxicity. Examination of the mechanism of action whereby these immunoconjugates induced cytotoxicity in MM cells demonstrated that treatment triggered G2/M cell cycle arrest, followed by apoptosis associated with cleavage of PARP and caspase-3, -8 and -9. Neither interleukin-6 nor insulin-like growth factor-I could overcome the apoptotic effect of these agents. The level of soluble (s)CD138 in the BM plasma from 15 MM patients was evaluated to determine the potential impact of sCD138 on immunoconjugate function. The sCD138 level in BM plasma was found to be significantly lower than that present in MM cell culture supernatants where potent in vitro cytotoxicity was observed, suggesting that sCD138 levels in MM patient BM plasma would not interfere with immunoconjugate activity. Because adhesion to bone marrow stromal cells (BMSCs) triggers cell adhesion mediated drug resistance to conventional therapies, we next examined the effects of the conjugates on MM cell growth in the context of BMSC. Co-culture of MM cells with BMSCs, which protects against dexamethasoneinduced death, had no impact on the cytotoxicity of the immunoconjugates. The in vivo efficacy of these immunoconjugates was also evaluated in SCID mice bearing established CD138-positive MM xenografts and in a SCID-human bone xenograft model of myeloma. Significant tumor growth delay or regressions were observed at immunoconjugate concentrations that were well tolerated in all models tested. The ability of these agents to mediate bystander killing of proximal CD138-negative cells was also evaluated. While nBT062-SPDB-DM4 was inactive against CD138-negative Namalwa cells cultured alone, significant killing of these CD138-negative cells by nBT062-SPDB-DM4 was observed when mixed with CD138-positive OPM2 cells. This bystander killing may contribute to the eradication of MM tumors by disrupting the tumor microenvironment and/or killing CD138-negative MM tumor cells, such as the putative CD138 negative myeloma stem cells. These studies demonstrate strong evidence of in vitro and in vivo selective cytotoxicity of these immunoconjugates and provide the preclinical framework supporting evaluation of nBT062-based immunoconjugates in clinical trials to improve patient outcome in MM.

Purified, Fermented, Extract of Triticum Aestivum Has Significant Independent Lymphomacidal Activity and Augments the Activity of Rituximab

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2857-2857
Author(s):  
Laura Newell ◽  
Joseph Tuscano ◽  
Robert o'Donnell ◽  
Yunpeng Ma
Keyword(s):  
Triticum Aestivum ◽  
T Cells ◽  
Cell Death ◽  
Cell Lines ◽  
Nude Mice ◽  
Mechanism Of Action ◽  
Wheat Germ ◽  
The United States

Abstract Abstract 2857 Background: Non-Hodgkin's lymphoma (NHL) affects over 400,000 people in the United States and its incidence increases with age. Treatment options include cytotoxic chemotherapy, which is often poorly tolerated by elderly patients, and monoclonal antibody (mAb) therapy. Nearly 70% of NHL patients eventually die of the disease. Development of effective alternate treatments with favorable toxicity profiles is necessary. Fermented wheat germ extract (FWGE) has shown anticancer potential in laboratory animals as well as in some small clinical studies; it is produced under GMP conditions in Europe and sold as Avemar™. The mechanism of action of FWGE is unclear, but is thought to involve metabolic pathways involved in tumor cell death. We examined the effects of FWGE on NHL and found significant lymphomacidal activity using in vitro and in vivo assays. We then further purified and characterized the active components of FWGE in order to develop a more potent form and to understand the mechanism of action, physiologic, and immunologic properties. Methods: FWGE was produced by fermenting purified wheat germ (Triticum aestivum) with Baker's yeast. The FWGE was further purified by removing insoluble material, precipitating proteins, freeze drying, fractionating with Sepharose and Sephadex columns, and then dialyzing to remove small molecules. The resultant fermented wheat germ proteins (FWGP) were assessed for in vitro cytotoxicity and pro-apoptotic activity using a panel of NHL cell lines. In vivo lymphomacidal activity was assessed in nude mice bearing Raji lymphoma xenografts. Mice were treated with increasing daily doses of FWGE by gastric lavage and compared to untreated controls as well as the commercially available fermented wheat germ product, Avemar. Results: In vitro killing assays with FWGE (regardless of the source) demonstrated lymphomacidal properties in three NHL cell lines (Jurkat, Raji, and Ramos). Pre-treatment of FWGE with heat or proteinase K reduced the lymphomacidal activity, suggesting that the active component was a protein. Nude mice bearing Raji lymphoma xenografts treated with FWGE confirmed the lymphomacidal properties of FGWE; there was no detectable toxicity as assessed by observation, mouse weight, or blood counts. The purified low molecular weight proteins (FWGP) also demonstrated lymphomacidal properties by cytotoxicity assays and murine NHL models, but at 1/1000th of the original dose. When FWGP was combined with rituximab, there was enhanced in vitro lymphomacidal activity, with over a 4000-fold reduction in the IC50. FWGP-induced NHL cell death was mediated by caspase-3-dependent apoptosis. FWGP augmented the host immune effector mechanisms, including ADCC and CDC, along with potent activation of NK-T cells (CD3/69/16), CD4+ T-cells and monocytes. Conclusions: FWGE can be easily produced and has cytotoxic effects in in vitro assays and in vivo. The purified FWGP are quantifiable, and are 10–1000 times more potent than FWGE. The mechanism of FWGP activity is based on direct pro-apoptotic effects as well as augmentation of host immune mediators. FWGP has activity against various subtypes of NHL. Studies are ongoing to further characterize the immune effects and anti-cancer properties of FWGP, as is planning for a human clinical trial +/− rituximab in patients with NHL. Disclosure: No relevant conflicts of interest to declare.

Modulation of enzymatic activity of Src-family kinases in bovine T cells transformed by Theileria parva

Parasitology ◽  
1998 ◽  
Vol 117 (2) ◽  
pp. 107-115 ◽  
Author(s):  
C. FICH ◽  
U. KLAUENBERG ◽  
B. FLEISCHER ◽  
B. M. BRÖKER
Keyword(s):  
T Cells ◽  
Enzymatic Activity ◽  
Cell Lines ◽  
Essential Role ◽  
Critical Role ◽  
Src Family Kinases ◽  
Theileria Parva ◽  
Transformation Mechanism ◽  
Transformed Cell Lines

After infection with sporozoites of the protozoon Theileria parva (Tp) bovine T cells are readily transformed to permanent growth in vivo and in vitro. Their transformed state depends on the constant presence of the parasite but membrane signals remain important. Non-receptor tyrosine kinases play a critical role in the transduction of membrane signals in haematopoietic cells. We have investigated Src-family kinases in bovine T cells transformed by Tp. The T cell receptor-associated tyrosine kinase p60fyn had high activity in all cell lines tested. In addition, weak phosphorylation of 2 novel bands was observed associated with Fyn. In contrast to Fyn, enzymatic activity of p56lck, which in T cells has an essential role in signalling, was low. Furthermore, 1 of 3 Tp transformed cell lines was completely devoid of p56lck indicating that the enzyme is not necessary for the Tp dependent growth of the T cells. In addition to p60fyn and p56lck weak enzymatic activity of 1 splice variant of p53/56lyn was observed after infection of T cells with Tp. These data show that growth transformation by Tp influences kinase activity in bovine T cells. However, they also prove that p56lck does not play an essential role in the transformation mechanism.

scholarly journals Growth-inhibitory Activity and Downregulation of the Class II Tumor-suppressor Gene H-rev107 in Tumor Cell Lines and Experimental Tumors

1997 ◽  
Vol 136 (4) ◽  
pp. 935-944 ◽  
Author(s):  
Christine Sers ◽  
Urban Emmenegger ◽  
Knut Husmann ◽  
Katharina Bucher ◽  
Ann-Catherine Andres ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Lines ◽  
Nude Mice ◽  
Class Ii ◽  
Tumor Formation ◽  
Pancreatic Tumors ◽  
Rat Tissues ◽  
Transformed Cell Lines

The H-rev107 gene is a new class II tumor suppressor, as defined by its reversible downregulation and growth-inhibiting capacity in HRAS transformed cell lines. Overexpression of the H-rev107 cDNA in HRAS-transformed ANR4 hepatoma cells or in FE-8 fibroblasts resulted in 75% reduction of colony formation. Cell populations of H-rev107 transfectants showed an attenuated tumor formation in nude mice. Cells explanted from tumors or maintained in cell culture for an extended period of time no longer exhibited detectable levels of the H-rev107 protein, suggesting strong selection against H-rev107 expression in vitro and in vivo. Expression of the truncated form of H-rev107 lacking the COOH-terminal membrane associated domain of 25 amino acids, had a weaker inhibitory effect on proliferation in vitro and was unable to attenuate tumor growth in nude mice. The H-rev107 mRNA is expressed in most adult rat tissues, and immunohistochemical analysis showed expression of the protein in differentiated epithelial cells of stomach, of colon and small intestine, in kidney, bladder, esophagus, and in tracheal and bronchial epithelium. H-rev107 gene transcription is downregulated in rat cell lines derived from liver, kidney, and pancreatic tumors and also in experimental mammary tumors expressing a RAS transgene. In colon carcinoma cell lines only minute amounts of protein were detectable. Thus, downregulation of H-rev107 expression may occur at the level of mRNA or protein.

Establishment and Characterization of Human Glioblastoma Cell Lines in vitro and their Xenografts in Nude Mice

1989 ◽  
Vol 12 (4) ◽  
pp. 169-174 ◽  
Author(s):  
H. Fischer ◽  
W.J. Zeller ◽  
K. Schwechheimer ◽  
K.-J. Hutter ◽  
B. Wowra ◽  
...  
Keyword(s):  
Cell Lines ◽  
Nude Mice ◽  
Glioblastoma Cell ◽  
Human Glioblastoma ◽  
Human Glioblastoma Cell ◽  
Glioblastoma Cell Lines
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