Inducible expression of the gap junction protein connexin43 decreases the neoplastic potential of HT-1080 human fibrosarcoma cells in vitro and in vivo

Angiogenesis inhibitors are a novel class of promising therapeutic agents for treating cancer. TNP-470, a systemic analogue of fumagillin, is an angiogenesis inhibitor capable of suppressing the tumorigenicity in several animal models even though the mechanisms of action have not been completely clarified. In the current study, we investigated the effects of TNP-470 on human fibrosarcoma cellsin vivoandin vitro. The administration of TNP-470 could suppress the tumorigenicity of HT1080 fibrosarcoma tumor. The conditioned medium from HT1080 fibrosarcoma cells treated with TNP-470 inhibited the proliferation and migration of human endothelial cell line, HUVEC and ECV304. The concentration of VEGF in the conditioned medium from HT1080 cells treated with TNP-470 was lower than that of the cells without TNP-470 treatment, indicating that TNP-470 downregulates the secretion of VEGF from HT1080 cells. These findings strongly suggest that the direct action of TNP-470 on sarcoma cells inhibits angiogenesis through the downregulation of VEGF secretion and this angiogenesis suppression resulted in the inhibition of tumorigenicity of HT1080 fibrosarcoma tumo.

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Sharp Wave-Like Activity in the Hippocampus In Vitro in Mice Lacking the Gap Junction Protein Connexin 36

Journal of Neurophysiology ◽

10.1152/jn.00549.2002 ◽

2003 ◽

Vol 89 (4) ◽

pp. 2046-2054 ◽

Cited By ~ 67

Author(s):

Isabel Pais ◽

Sheriar G. Hormuzdi ◽

Hannah Monyer ◽

Roger D. Traub ◽

Ian C. Wood ◽

...

Keyword(s):

Gap Junctions ◽

Gap Junction ◽

Frequency Oscillation ◽

Connexin 36 ◽

Sharp Wave ◽

Bath Application ◽

Junction Protein ◽

Gap Junction Protein ◽

Electrical Signaling

Bath application of kainate (100–300 nM) induced a persistent gamma-frequency (30–80 Hz) oscillation that could be recorded in stratum radiatum of the CA3 region in vitro. We have previously described that in knockout mice lacking the gap junction protein connexin 36 (Cx36KO), γ-frequency oscillations are reduced but still present. We now demonstrate that in the Cx36KO mice, but not in wild-type (WT), large population field excitatory postsynaptic potentials, or sharp wave-burst discharges, also occurred during the on-going γ-frequency oscillation. These spontaneous burst discharges were not seen in WT mice. Burst discharges in the Cx36KO mice occurred with a mean frequency of 0.23 ± 0.11 Hz and were accompanied by a series of fast (approximately 60–115 Hz) population spikes or “ripple” oscillations in many recordings. Intracellular recordings from CA3 pyramidal cells showed that the burst discharges consisted of a depolarizing response and presumed coupling potentials (spikelets) could occasionally be seen either before or during the burst discharge. The burst discharges occurring in Cx36KO mice were sensitive to gap junctions blockers as they were fully abolished by carbenoxolone (200 μM). In control mice we made several attempts to replicate this pattern of sharp wave activity/ripples occurring with the on-going kainate-evoked γ-frequency oscillation by manipulating synaptic and electrical signaling. Partial disruption of inhibition, in control slices, by bath application of the γ-aminobutyric acid-A (GABAA) receptor antagonist bicuculline (1–4 μM) completely abolished all γ-frequency activity before any burst discharges occurred. Increasing the number of open gap junctions in control slices by using trimethylamine (TMA; 2–10 mM), in conjunction with kainate, failed to elicit any sharp wave bursts or fast ripples. However, bath application of the potassium channel blocker 4-aminopyridine (4-AP; 20–80 μM) produced a pattern of activity in control mice (13/16 slices), consisting of burst discharges occurring in conjunction with kainate-evoked γ-frequency oscillations, that was similar to that seen in Cx36KO mice. In a few cases ( n = 9) the burst discharges were accompanied by fast ripple oscillations. Carbenoxolone also fully blocked the 4-AP-evoked burst discharges ( n = 5). Our results show that disruption of electrical signaling in the interneuronal network can, in the presence of kainate, lead to the spontaneous generation of sharp wave/ripple activity similar to that observed in vivo. This suggests a complex role for electrically coupled interneurons in the generation of hippocampal network activity.

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Five-hour half-life of mouse liver gap-junction protein.

The Journal of Cell Biology ◽

10.1083/jcb.90.2.521 ◽

1981 ◽

Vol 90 (2) ◽

pp. 521-526 ◽

Cited By ~ 206

Author(s):

R F Fallon ◽

D A Goodenough

Keyword(s):

Gap Junctions ◽

Gap Junction ◽

Half Life ◽

Mouse Liver ◽

Polyacrylamide Gels ◽

Junction Protein ◽

Gap Junction Protein ◽

Polypeptide Band

The half-life of a gap-junction polypeptide band migrating at 21,000 Mr on SDS polyacrylamide gels isolated from mouse liver is measured to be 5 h. Two low-molecular wight bands, probably related to the 21,000 Mr material by proteolysis, have measured half-lives of 4.6 and 5.2 h. Gap junctions are labeled in vivo using the 14C-bicarbonate labeling procedure, followed by quantitative fluorography.

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Connexin 43 confers chemoresistance through activating PI3K

Oncogenesis ◽

10.1038/s41389-022-00378-7 ◽

2022 ◽

Vol 11 (1) ◽

Author(s):

Kevin J. Pridham ◽

Farah Shah ◽

Kasen R. Hutchings ◽

Kevin L. Sheng ◽

Sujuan Guo ◽

...

Keyword(s):

Poor Prognosis ◽

Connexin 43 ◽

Therapeutic Potential ◽

Mrna Levels ◽

Phosphatidylinositol 3 Kinase ◽

Junction Protein ◽

Gap Junction Protein ◽

Cx43 Mrna

AbstractCircumventing chemoresistance is crucial for effectively treating cancer including glioblastoma, a lethal brain cancer. The gap junction protein connexin 43 (Cx43) renders glioblastoma resistant to chemotherapy; however, targeting Cx43 is difficult because mechanisms underlying Cx43-mediated chemoresistance remain elusive. Here we report that Cx43, but not other connexins, is highly expressed in a subpopulation of glioblastoma and Cx43 mRNA levels strongly correlate with poor prognosis and chemoresistance in this population, making Cx43 the prime therapeutic target among all connexins. Depleting Cx43 or treating cells with αCT1–a Cx43 peptide inhibitor that sensitizes glioblastoma to the chemotherapy temozolomide–inactivates phosphatidylinositol-3 kinase (PI3K), whereas overexpression of Cx43 activates this signaling. Moreover, αCT1-induced chemo-sensitization is counteracted by a PI3K active mutant. Further research reveals that αCT1 inactivates PI3K without blocking the release of PI3K-activating molecules from membrane channels and that Cx43 selectively binds to the PI3K catalytic subunit β (PIK3CB, also called PI3Kβ or p110β), suggesting that Cx43 activates PIK3CB/p110β independent of its channel functions. To explore the therapeutic potential of simultaneously targeting Cx43 and PIK3CB/p110β, αCT1 is combined with TGX-221 or GSK2636771, two PIK3CB/p110β-selective inhibitors. These two different treatments synergistically inactivate PI3K and sensitize glioblastoma cells to temozolomide in vitro and in vivo. Our study has revealed novel mechanistic insights into Cx43/PI3K-mediated temozolomide resistance in glioblastoma and demonstrated that targeting Cx43 and PIK3CB/p110β together is an effective therapeutic approach for overcoming chemoresistance.

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Combination treatment with arsenic trioxide and irradiation enhances cell-killing effects in human fibrosarcoma cells in vitro and in vivo through induction of both autophagy and apoptosis

Autophagy ◽

10.4161/auto.6.3.11229 ◽

2010 ◽

Vol 6 (3) ◽

pp. 353-365 ◽

Cited By ~ 57

Author(s):

Hui-Wen Chiu ◽

Jing-Hua Lin ◽

Yi-An Chen ◽

Sheng-Yow Ho ◽

Ying-Jan Wang

Keyword(s):

Arsenic Trioxide ◽

Combination Treatment ◽

Cell Killing ◽

Fibrosarcoma Cells ◽

Human Fibrosarcoma Cells

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Stanniocalcin 1 and 2 are secreted as phosphoproteins from human fibrosarcoma cells

Biochemical Journal ◽

10.1042/bj3500453 ◽

2000 ◽

Vol 350 (2) ◽

pp. 453-461 ◽

Cited By ~ 34

Author(s):

Derek A. JELLINEK ◽

Andy C. CHANG ◽

Martin R. LARSEN ◽

Xin WANG ◽

Phillip J. ROBINSON ◽

...

Keyword(s):

Protein Kinase ◽

Mammalian Cells ◽

Phosphorylation Site ◽

Mrna Levels ◽

Intact Cells ◽

Fibrosarcoma Cells ◽

Ht1080 Cells ◽

Human Fibrosarcoma Cells

Stanniocalcin 1 (STC1) and stanniocalcin 2 (STC2) are two recently identified mammalian peptide hormones. STC1 plays a role in calcium and phosphate homoeostasis, while the role of STC2 is unknown. We examined a human fibrosarcoma cell line, HT1080, that has high steady-state STC1 and STC2 mRNA levels, to determine whether these proteins are secreted. Following incubation of HT1080 cells with 32P, labelled STC1 and STC2 were found to be secreted into the medium. STC1 was phosphorylated in vitro by protein kinase C (PKC). In vitro and in vivo phosphorylation both occurred exclusively on serine and the phosphopeptide maps were similar, suggesting that PKC might be the in vivo kinase. STC2 was phosphorylated in vitro by casein kinase II (CK2), in vitro and in vivo phosphorylation were exclusively on serine and the phosphopeptide maps were indistinguishable. Phosphorylation of STC2 in intact cells resulted from the action of an ecto-protein kinase, since exogenous STC2 was phosphorylated by HT1080 cells and no phosphorylated STC2 was detectable inside the cells. The ectokinase activity was abolished by heparin and GTP could substitute for ATP as the phosphate donor, indicative of an ecto-CK2-like activity. The in vitro CK2 phosphorylation site was shown by matrix-assisted laser-desorption ionization–time-of-flight MS to be a single serine located between Ser-285 and Ser-298 in the C-terminal region of STC2. This is the first report of the secretion of STC1 or STC2 from mammalian cells. We conclude that these human fibrosarcoma cells express both STC1 and STC2 as secreted phosphoproteins in vivo, with STC2 being phosphorylated by an ecto-CK2-like enzyme.

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