NIH 3T3 Mouse Fibroblast Cells Research Articles

Properties of a novel quaterpolymer P(3HB/4HB/3HV/3HHx)

Cupriavidus eutrophus В10646 was used to synthesize a series of polyhydroxyalkanoate (PHA) quaterpolymers composed of the short-chain-length 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), and 3-hydroxyvalerate (3HV) and the medium-chain-length 3-hydroxyhexanoate (3HHx). The molar fraction of 3HB in the quaterpolymers varied between 63.5 and 93.1 mol.%, 3HV – between 1.1 and 24.6 mol.%, 4HB – between 2.4 and 15.6 mol.%, and 3HHx – between 0.4 and 4.8 mol.%. The properties of PHA quaterpolymers were significantly different from those of the P(3HB) homopolymer: they had much lower degrees of crystallinity (up to 30–45%), and lower melting points and thermal decomposition temperatures, with the interval between these temperatures remaining practically unchanged. Films prepared from PHA quaterpolymers were rougher and more porous than P(3HB) films; they showed higher values of elongation at break (up to 6–113%), i.e. were more elastic. Films prepared from PHA quaterpolymers were biocompatible and had no toxic effect on mouse fibroblast NIH 3T3 cells.

BET bromodomain inhibitors suppress EWS-FLI1-dependent transcription and the IGF1 autocrine mechanism in Ewing sarcoma.

Ewing sarcoma is driven by characteristic chromosomal translocations between the EWSR1 gene with genes encoding ETS family transcription factors (EWS-ETS), most commonly FLI1. However, direct pharmacological inhibition of transcription factors like EWS-FLI1 remains largely unsuccessful. Active gene transcription requires orchestrated actions of many epigenetic regulators, such as the bromodomain and extra-terminal domain (BET) family proteins. Emerging BET bromodomain inhibitors have exhibited promising antineoplastic activities via suppression of oncogenic transcription factors in various cancers. We reasoned that EWS-FLI1-mediated transcription activation might be susceptible to BET inhibition. In this study, we demonstrated that small molecule BET bromodomain inhibitors repressed EWS-FLI1-driven gene signatures and downregulated important target genes. However, expression of EWS-FLI1 was not significantly affected. Repression of autocrine IGF1 by BET inhibitors led to significant inhibition of the IGF1R/AKT pathway critical to Ewing sarcoma cell proliferation and survival. Consistently, BET inhibitors impaired viability and clonogenic survival of Ewing sarcoma cell lines and blocked EWS-FLI1-induced transformation of mouse NIH3T3 fibroblast cells. Selective depletion of individual BET genes partially phenocopied the actions of BET inhibitors. Finally, the prototypical BET inhibitor, JQ1, significantly repressed Ewing sarcoma xenograft tumor growth. These findings suggest therapeutic potential of BET inhibitors in Ewing sarcoma and highlight an emerging paradigm of using epigenetic agents to treat cancers driven by fusion transcription factors.

Identification of cytoprotective constituents of the flower buds of Tussilago farfara against glucose oxidase-induced oxidative stress in mouse fibroblast NIH3T3 cells and human keratinocyte HaCaT cells

A new cytoprotective compound, 1-[(4S)-3,4-dihydro-4-hydroxy-2,2-dimethyl-2H-1-benzopyran-6-yl]-ethanone (1) was isolated from the flower buds of Tussilago farfara L. (Compositae), together with eight known compounds, 3,4-dicaffeoyl isoquinic acid (2), trans-cinnamic acid (3), 4-hydroxyacetophenone (4), 4,5-dicaffeoylquinic acid methyl ester (5), 3,5-dicaffeoylquinic acid methyl ester (6), 4-hydroxybenzoic acid (7), isoquercetrin (8), and ligucyperonol (9). Compounds 2-4 were found in this plant for the first time. The isolates 1-9, were tested for their cytoprotective activities against glucose oxidase-induced oxidative stress in mouse fibroblast NIH3T3 cells and human keratinocyte HaCaT cells. Among them, 1 and 3 showed significant cytoprotective activities as determined by MTT assay and lactate dehydrogenase leakage, indicating their possibility as the potent cytoprotective agents. The structure of 1 was determined by spectroscopic data analysis including 1D- and 2D-NMR experiments, and its absolute configuration was elucidated by a circular dichroism.

Clean localization super-resolution microscopy for 3D biological imaging

We propose clean localization microscopy (a variant of fPALM) using a molecule filtering technique. Localization imaging involves acquiring a large number of images containing single molecule signatures followed by one-to-one mapping to render a super-resolution image. In principle, this process can be repeated for other z-planes to construct a 3D image. But, single molecules observed from off-focal planes result in false representation of their presence in the focal plane, resulting in incorrect quantification and analysis. We overcome this with a single molecule filtering technique that imposes constraints on the diffraction limited spot size of single molecules in the image plane. Calibration with sub-diffraction size beads puts a natural cutoff on the actual diffraction-limited size of single molecules in the focal plane. This helps in distinguishing beads present in the focal plane from those in the off-focal planes thereby providing an estimate of the single molecules in the focal plane. We study the distribution of actin (labeled with a photoactivatable CAGE 552 dye) in NIH 3T3 mouse fibroblast cells.

A simple aza-crown ether containing an anthraquinone fluorophore for the selective detection of Mg(II) in living cells

A new aza-crown ether anthraquinone-coupled chemosensor (2) has been synthesized and employed for the selective fluorescence detection of Mg(II) in the nanomolar range with limited interference by Ca(II) ion. The binding stoichiometry for the chemosensor with Mg(II) is found to be 2:1, and the binding constant was determined to be 2.9×109 M−2 and 5.1×109 M−2 in acetonitrile and DMSO, respectively. The chemosensor also shows negligible fluorescence enhancement from other alkali, alkaline earth, and transition metals. Chemosensor 2 has been used in the bioimaging of Mg(II) in embryonic mouse fibroblast NIH3T3 cells at both native concentrations and cells incubated with excess Mg(II). The chemosensor can access Mg(II) ions in both the cytoplasm and nucleus of the cells. The corrected total cell fluorescence is also calculated to confirm the uptake of the chemosensor in living cells.

Improved cellular response of ion modified poly(lactic acid-co-glycolic acid) substrates for mouse fibroblast cells

In this report, the effects of argon (Ar) ion irradiation on poly(lactic acid-co-glycolic acid) (PLGA) substrates on biocompatibility were studied. PLGA scaffold substrates were prepared by spin coating glass surfaces with PLGA dissolved in anhydrous chloroform. Previously, we showed that surface modifications of PLGA films using ion irradiation modulate the inherent hydrophobicity of PLGA surface. Here we show that with increasing ion dose (1×1012 to 1×1014ions/cm2), hydrophobicity and surface roughness decreased. Biocompatibility for NIH3T3 mouse fibroblast cells was increased by argon irradiation of PLGA substrates. On unirradiated PLGA films, fibroblasts had a longer doubling time and cell densities were 52% lower than controls after 48h in vitro. Argon irradiated PLGA substrates supported growth rates similar to control. Despite differences in cell cycle kinetics, there was no detectible cytotoxicity observed on any substrate. This demonstrates that argon ion irradiation can be used to tune the surface microstructure and generate substrates that are more compatible for the cell growth and proliferation.

Composite wound dressings of pectin and gelatin with aloe vera and curcumin as bioactive agents

Aloe vera and curcumin loaded oxidized pectin–gelatin (OP-Gel) matrices were used as antimicrobial finishes on nonwoven cotton fabrics to produce composite wound care devices. The drug release characteristics of the biocomposite dressings indicated that curcumin is released through a biphasic mechanism – erosion of the polymeric matrix, followed by diffusion, while aloe vera is released upon leaching of the polymeric matrix. A 50/50 composition of aloe vera/curcumin was used to fabricate OP-Gel-Aloe Curcumin dressings. However, contrary to our expectations, OP-Gel-Aloe Curcumin dressings exhibited lesser antimicrobial activity compared to OP-Gel-Aloe and OP-Gel-Curcumin dressings. The cytocompatibility of the fabricated dressings was evaluated using NIH3T3 mouse fibroblast cells. OP-Gel-Aloe treated fibroblasts had the highest viability, with the matrices providing a substrate for good cell attachment and proliferation. On the other hand, OP-Gel-Curcumin and OP-Gel-Aloe Curcumin seemed to have induced apoptosis in NIH3T3 cells. In vivo wound healing analysis was carried out using an excisional splint wound model on C57BL/6J mice. OP-Gel-Aloe treated wounds exhibited very rapid healing with 80% of the wound healing in just 8 days. Furthermore, aloe vera exerted a strong anti-inflammatory effect and prominent scar prevention. Histological examination revealed that an ordered collagen formation and neovascularization could be observed along with migration of nuclei. Therefore, OP-Gel-Aloe biocomposite dressings are proposed as viable materials for effective wound management.

Laser processing of polymer constructs from poly(3-hydroxybutyrate)

CO2 laser radiation was used to process poly(3-hydroxybutyrate) constructs – films and 3D pressed plates. Laser processing increased the biocompatibility of unperforated films treated with moderate uniform radiation, as estimated by the number and degree of adhesion of NIH 3T3 mouse fibroblast cells. The biocompatibility of perforated films modified in the pulsed mode did not change significantly. At the same time, pulsed laser processing of the 3D plates produced perforated scaffolds with improved mechanical properties and high biocompatibility with bone marrow-derived multipotent, mesenchymal stem cells, which show great promise for bone regeneration.

Abstract 3545: Stromal selective targeting by uPAR retargeted oncolytic measles virus inhibits breast cancer progression

Abstract Stromal cells in the tumor microenvironment play an important role in breast cancer progression. Strategies directed against stromal cell components have been investigated in the preclinical and clinical setting. However, few studies have focused on stromal targeting by oncolytic viruses. The urokinase receptor (uPAR) is overexpressed in tumor and stromal cells, and plays a critical role in tumor progression. The aim of this study is to evaluate the antitumor effects of stromal retargeted oncolytic measles virus via urokinase receptor. We rescued and characterized fully retargeted oncolytic measles viruses against human (MV-h-uPA) and murine (MV-m-uPA) urokinase receptor (uPAR). In vitro, MV-m-uPA and MV-h-uPA infected and induced cytotoxicity to human (MDA-MB231, T47D and MCF-7) and murine (4T1) mammary cancer cells in a species specific manner. Efficient MV-m-uPA infection and replication were demonstrated in murine NIH-3T3 mouse fibroblast cells and MS1 murine endothelial cells. Selective infection of murine fibroblasts by the murine uPAR specific MV-m-uPA led to significant inhibition of human breast cancer proliferation in an in vitro 3-D collagen co-culture model of tumor-stromal interactions. To further validate the potential of stromal uPAR targeting as a therapeutic strategy, the effects of MV-uPA were assessed in a human breast cancer xenograft model (MDA-MD231), where tumor cells express human uPAR (target of MV-h-uPA) and the host stroma expresses murine uPAR (target of MV-m-uPA). Tumor bearing mice were treated with MV-h-uPA, MV-m-uPA or combination (MV-h-uPA and MV-m-uPA) by IV administration (3 doses). Surprisingly, MV-m-uPA (selectively targeting mouse uPAR) was associated with a measurable delay in tumor progression while on treatment, as well as prolongation in survival. Combination therapy (MV-m-uPA and MV-h-uPA) was associated with the best antitumor effects in vivo. To further characterize the in vivo effects of stromal uPAR targeting, repeat experiments were performed where tumor bearing mice received prolonged treatment with MV-m-uPA (12 treatments). This approach was shown to be safe and resulted in a significant delay in tumor progression and marked prolongation of survival. These effects were associated with the identification of viable viral particles and viral RNA from MDA-MB231 tumor xenograft treated with MV-m-uPA. In conclusion, our results demonstrate for the first time that oncolytic viral targeting of tumor stroma is feasible and associated with in vivo antitumor effects. These findings further validate the critical role of stromal uPAR in cancer progression. Citation Format: Yuqi Jing, Marcela Toro Bejarano, Krisztina Kovacs, Jaime Merchan. Stromal selective targeting by uPAR retargeted oncolytic measles virus inhibits breast cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3545. doi:10.1158/1538-7445.AM2015-3545

A novel isoflavone from Leiophyllum buxifolium (Ericaceae) and its antiproliferative effect

A new C-methyl-isoflavone, isosideroxylin 1, and a known C-methyl-flavone, sideroxylin 2, were isolated from the EtOAc extract of the leaves of Leiophyllum buxifolium. HR-ASAP-MS and 1D and 2D NMR spectroscopic data were used to determine the structure of 1. The two compounds were evaluated for their antiproliferative effects against ER-MDA-MB-231 and ER+MCF-7 breast cancer cell lines and the NIH3T3 mouse fibroblast cell line with the sulforhodamine B assay. Compound 2 showed moderate antiproliferative activity on both cancer cell lines, while compound 1 displayed selective antiproliferative effect against MDA-MB-231 cells only, which supports the hypothesis of phytoestrogenic activity of 1. Neither compound was inhibitory to the NIH3T3 mouse fibroblasts at the concentrations examined. Since 1 and 2 share the A ring substitution pattern, we concluded the 5-hydroxy-7-methoxy-6,8-dimethyl substitution pattern on the A ring is responsible for the antiproliferative activity. The presence of a C-methyl-isoflavone in the Ericaceae supports the presence of a isoflavone synthesis pathway in this family.

Silica/quercetin sol–gel hybrids as antioxidant dental implant materials

The development of biomaterials with intrinsic antioxidant properties could represent a valuable strategy for preventing the onset of peri-implant diseases. In this context, quercetin, a naturally occurring flavonoid, has been entrapped at different weight percentages in a silica-based inorganic material by a sol–gel route. The establishment of hydrogen bond interactions between the flavonol and the solid matrix was ascertained by Fourier transform infrared spectroscopy. This technique also evidenced changes in the stretching frequencies of the quercetin dienonic moiety, suggesting that the formation of a secondary product occurs. Scanning electron microscopy was applied to detect the morphology of the synthesized materials. Their bioactivity was shown by the formation of a hydroxyapatite layer on sample surface soaked in a fluid that simulates the composition of human blood plasma. When the potential release of flavonol was determined by liquid chromatography coupled with ultraviolet and electrospray ionization tandem mass spectrometry techniques, the eluates displayed a retention time that was 0.5 min less than quercetin. Collision-activated dissociation mass spectrometry and untraviolet-visible spectroscopy were in accordance with the release of a quercetin derivative. The antiradical properties of the investigated systems were evaluated by DPPH and ABTS methods, whereas the 2,7-dichlorofluorescein diacetate assay highlighted their ability to inhibit the H2O2-induced intracellular production of reactive oxygen species in NIH-3T3 mouse fibroblast cells. Data obtained, along with data gathered from the MTT cytotoxicity test, revealed that the materials that entrapped the highest amount of quercetin showed notable antioxidant effectiveness.

An Isoflavone from Leiophyllum buxifolium and Its Antiproliferative Effect.

A new C-methylisoflavone, isosideroxylin (1), and a known C-methylflavone, sideroxylin (2), were isolated from the EtOAc extract of the leaves of Leiophyllum buxifolium. The two compounds were evaluated with the sulforhodamine B assay for their antiproliferative effects against ER(-) MDA-MB-231 and ER(+) MCF-7 breast cancer cell lines and the NIH3T3 mouse fibroblast cell line. Isosideroxylin (1) displayed a selective antiproliferative effect against MDA-MB-231 cells.

Cloning and Stable Expression of cDNA Coding For Platelet Endothelial Cell Adhesion Molecule -1 (PECAM-1, CD31) in NIH-3T3 Cell Line.

PECAM-1 (CD31) is a glycoprotein expressed on endothelial and bone marrow precursor cells. It plays important roles in angiogenesis, maintenance and integration of the cytoskeleton and direction of leukocytes to the site of inflammation. We aimed to clone the cDNA coding for human CD31 from KG1a for further subcloning and expression in NIH-3T3 mouse cell line. CD31 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. 2235 bp specific band was aligned completely to human CD31 reference sequence in NCBI database. Transient and stable expression of human CD31 on transfected NIH-3T3 mouse fibroblast cells was achieved (23% and 96%, respectively) as shown by flow cytometry. Due to murine origin of NIH-3T3 cell line, CD31-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD31, with no need for purification of recombinant proteins.

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line.

Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems.

Fluorescent cadmium telluride quantum dots embedded chitosan nanoparticles: a stable, biocompatible preparation for bio-imaging

Fluorescent cadmium telluride quantum dots (CdTe QDs) are an optically attractive option for bioimaging, but are known to display high cytotoxicity. Nanoparticles synthesized from chitosan, a natural biopolymer of β 1-4 linked glucosamine, display good biocompatibility and cellular uptake. A facile, green synthetic strategy has been developed to embed green fluorescent cadmium telluride quantum dots (CdTe QDs) in biocompatible CNPs to obtain a safer preparation than ‘as is’ QDs. High-resolution transmission electron microscopy showed the crystal lattice corresponding to CdTe QDs embedded in CNPs while thermogravimetry confirmed their polymeric composition. Electrostatic interactions between thiol-capped QDs (4 nm, −57 mV) and CNPs (~300 nm, +38 mV) generated CdTe QDs-embedded CNPs that were stable up to three months. Further, viability of NIH3T3 mouse fibroblast cells in vitro increased in presence of QDs-embedded CNPs as compared to bare QDs. At the highest concentration (10 μg/ml), the former shows 34 and 39% increase in viability at 24 and 48 h, respectively, as compared to the latter. This shows that chitosan nanoparticles do not release the QDs up to 48 h and do not cause extended toxicity. Furthermore, hydrolytic enzymes such as lysozyme and chitinase did not degrade chitosan nanoparticles. Moreover, QDs-embedded CNPs show enhanced internalization in NIH3T3 cells as compared to bare QDs. This method offers ease of synthesis and handling of stable, luminescent, biocompatible CdTe QDs-embedded CNPs with a favorable toxicity profile and better cellular uptake with potential for bioimaging and targeted detection of cellular components.

Transformation of NIH3T3 mouse fibroblast cells by MUC16 mucin (CA125) is driven by its cytoplasmic tail.

MUC16 (CA125) is a transmembrane mucin that contributes to the progression of epithelial ovarian cancer (EOC). Expression of MUC16 is not detectable in the epithelial surface of normal ovaries. MUC16 expression is, however, common in serous EOC as well as in metastatic and recurrent tumors. Despite these observations, its contribution to the development of EOC is unknown. We stably expressed either empty vector, MUC16 C-terminal domain (MUC16 CTD) or MUC16 TMU (a construct that lacks the cytoplasmic tail) in NIH3T3 mouse fibroblast cells. In this study, we provide evidence for the role of MUC16 CTD in oncogenic transformation. We show that ectopic expression of MUC16 CTD enhances the growth of NIH3T3 cells under normal and low serum conditions, and promotes anchorage-dependent colony formation. The deletion of the cytoplasmic tail abrogated these effects. MUC16 CTD expression in NIH3T3 cells also enhances the formation of colony in soft agar as compared to MUC16 TMU. MUC16 CTD expression enhances tumor formation in nude mice. Our findings provide the first evidence that MUC16 induces the transformation of NIH3T3 cells and indicate that MUC16 functions as an oncogene. Furthermore, our data suggest that the cytoplasmic tail is critical for MUC16 oncogenic properties.

Effects of fasudil on bleomycin-induced pulmonary fibrosis in mice and on the biological behaviors in NIH3T3 mouse fibroblast cell line

To determine the beneficial effects and mechanisms of fasudil, a selective ROCK inhibitor, on bleomycin-induced pulmonary fibrosis in mice and to determine the effects and mechanisms of fasudil on the biological behaviors in NIH3T3 mouse fibroblast cell line. The BPF model was induced by a single dosage of 2.5 mg/kg bleomycin intratracheal injection in mice and fasudil intraperitoneal injection was given to the mice. The fibrosis degree was determined pathologically by using the Ashcroft scoring method and biochemically by hydroxyproline assay in lung tissue. NIH3T3 mouse fibroblast cell line was cultured in vitro and fasudil was given to the cell. The proliferation activity in NIH3T3 cells were detected by MTT assay and flat colony forming experiment. The migration activity in NIH3T3 cells were detected by scratch test and transwell chamber experiment. The expression of CyclinD1, MMP2 and TIMP1 mRNA in NIH3T3 cells was detected by RT-PCR. The expression of CyclinD1, MMP2 and TIMP1 protein and the level of MYPT1 phosphorylation in NIH3T3 cells was detected by Western blot. Compare to the mice administrated by bleomycin, the Ashcroft score and hydroxyproline content were significantly decreased in the mice administered fasudil. Administration of fasudil can reduce the ability of proliferation and migration in a dose-dependent manner in NIH3T3 cells. The effect of fasudil was possibly related to increase the production of TIMP1 and decrease the production of CyclinD1 and MMP2. Administration of fasudil can attenuate pulmonary fibrosis both in vivo and in vitro. These findings suggest that fasudil may be a potential therapeutic candidate for the treatment of pulmonary fibrosis.

The expression of pluripotency genes and neuronal markers after neurodifferentiation in fibroblasts co-cultured with human umbilical cord blood mononuclear cells.

Human umbilical cord blood is an attractive source of stem cells; however, it has a heterogeneous cell population with few mesenchymal stem cells. Cell reprogramming induced by different methodologies can confer pluripotency to differentiated adult cells. The objective of this study was to evaluate the reprogramming of fibroblasts and their subsequent neural differentiation after co-culture with umbilical cord blood mononuclear cells. Cells were obtained from four human umbilical cords. The mononuclear cells were cultured for 7 d and subsequently co-cultured with mouse fibroblast NIH-3T3 cells for 6 d. The pluripotency of the cells was evaluated by RT-PCR using primers specific for pluripotency marker genes. The pluripotency was also confirmed by adipogenic and osteogenic differentiation. Neural differentiation of the reprogrammed cells was evaluated by immunofluorescence. All co-cultured cells showed adipogenic and osteogenic differentiation capacity. After co-cultivation, cells expressed the pluripotency gene KLF4. Statistically significant differences in cell area, diameter, optical density, and fractal dimension were observed by confocal microscopy in the neurally differentiated cells. Contact in the form of co-cultivation of fibroblasts with umbilical cord blood mononuclear fraction for 6 d promoted the reprogramming of these cells, allowing the later induction of neural differentiation.

Translational assessment of mitochondrial dysfunction of pancreatic cancer from in vitro gene microarray and animal efficacy studies, to early clinical studies, via the novel tumor-specific anti-mitochondrial agent, CPI-613.

Via genetic alterations, malignant transformation and proliferation are associated with extensive alterations of mitochondrial energy metabolism of tumor cells. Thus, inhibition of the altered form of mitochondrial energy metabolism of tumor cells may be an effective therapy for cancers. This study performed translational assessment of mitochondrial dysfunction of pancreatic cancer from in vitro gene microarray and animal efficacy studies, to early clinical studies, via the novel tumor-specific anti-mitochondrial agent, CPI-613. The gene profiles of BxPC-3 human pancreatic tumor cells and non-transformed NIH-3T3 mouse fibroblast cells (negative control), after CPI-613 or sham treatment, were assessed and compared using microarray technique. The anti-cancer efficacies of CPI-613 and Gemcitabine were assessed and compared in mice with xenograft from inoculation of BxPC-3 human pancreatic tumor cells, based on the degree of tumor growth inhibition and prolongation of survival when compared to vehicle treatment. The anti-cancer activities, according to overall survival (OS), of CPI-613 alone and in combination with Gemcitabine were assessed in patients with Stage IV pancreatic cancer. Microarray studies indicated that CPI-613 down-regulated the expression of Cyclin D3, E1, E2, F, A2, B1 and CDK2 genes of BxPC-3 pancreatic cancer cells but not non-transformed NIH-3T3 mouse fibroblast cells (negative control). In mice with pancreatic carcinoma xenografts, four weekly intraperitoneal injections of either CPI-613 (25 mg/kg/administration) or Gemcitabine (50 mg/kg/administration) inhibited tumor growth and prolonged survival when compared to vehicle treatment. The degree of tumor growth inhibition was ~2×, and prolongation of survival was ~4×, greater with CPI-613 treatment than with Gemcitabine treatment. In patients with Stage IV advanced pancreatic cancer, CPI-613 at 420-1,300 mg/m(2), given twice weekly for three weeks followed by a week of rest (i.e., 3-week-on-1-week-off) as monotherapy, provided median OS of 15 months in three patients. CPI-613 at 150-320 mg/m(2) given twice weekly on the 3-week-on-1-week-off dosing schedule, coinciding with Gemcitabine (1,000 mg/m(2)) given once weekly on the 3-week-on-1-week-off dosing schedule, provided median OS of 17.8 months in four patients. These median OS values from CPI-613 monotherapy and CPI-613 + Gemcitabine treatment tend to be longer than those in patients treated with Abraxane + Gemcitabine combination or FOLFININOX (median OS ~12 months). The dysfunctional mitochondria of pancreatic cancer cells was translationable from in vitro gene alteration and animal tumor model studies to patients with advanced Stage IV pancreatic cancer, as reflected by the anti-cancer activities of the tumor-specific anti-mitochondrial agent, CPI-613, in these studies.

A Novel Mechanism by Which Tissue Transglutaminase Activates Signaling Events That Promote Cell Survival

Tissue transglutaminase (tTG) functions as a GTPase and an acyl transferase that catalyzes the formation of protein cross-links. tTG expression is frequently up-regulated in human cancer, where it has been implicated in various aspects of cancer progression, including cell survival and chemo-resistance. However, the extent to which tTG cooperates with other proteins within the context of a cancer cell, versus its intrinsic ability to confer transformed characteristics to cells, is poorly understood. To address this question, we asked what effect the ectopic expression of tTG in a non-transformed cellular background would have on the behavior of the cells. Using NIH3T3 fibroblasts stably expressing a Myc-tagged form of tTG, we found that tTG strongly protected these cells from serum starvation-induced apoptosis and triggered the activation of the PI3-kinase/mTOR Complex 1 (mTORC1)/p70 S6-kinase pathway. We determined that tTG forms a complex with the non-receptor tyrosine kinase c-Src and PI3-kinase, and that treating cells with inhibitors to block tTG function (monodansylcadaverine; MDC) or c-Src kinase activity (PP2) disrupted the formation of this complex, and prevented tTG from activating the PI3-kinase pathway. Moreover, treatment of fibroblasts over-expressing tTG with PP2, or with inhibitors that inactivate components of the PI3-kinase pathway, including PI3-kinase (LY294002) and mTORC1 (rapamycin), ablated the tTG-promoted survival of the cells. These findings demonstrate that tTG has an intrinsic capability to stimulate cell survival through a novel mechanism that activates PI3-kinase signaling events, thus highlighting tTG as a potential target for the treatment of human cancer.