- Library Home /
- Search Collections /
- Open Collections /
- Browse Collections /
- UBC Theses and Dissertations /
- Dihydromotuporamine C increases Na⁺/H⁺exchange activity...
Open Collections
UBC Theses and Dissertations
UBC Theses and Dissertations
Dihydromotuporamine C increases Na⁺/H⁺exchange activity via a Rho-kinase-dependent pathway and induces disruption of intercellular junctions in mammary epithelia Sinotte, Ryan Richard
Abstract
Invasion and angiogenesis are two major steps in the progression towards metastatic cancer. Dihydromotuporamine C (dh-MotC) inhibits both of these processes. To better characterize the mechanism of dh-MotC action, I examined the compound's effects on Na+/H+ exchange (NHE) activity and normal mammary epithelial cell viability and junctional architecture. In contrast to squalamine, an anti-angiogenic compound that inhibits Na+/H+ exchanger isoform 3 (NHE3), dh-MotC (5 or 10μM) increased the rate of recovery from an imposed acid load in nominally bicarbonate free conditions. This increased rate of recovery was inhibited by HOE694, a selective NHE inhibitor, and the Rho-kinase (ROCK) inhibitor Y-27632. Dh-MotC also induced an upward shift in steady state intracellular pH (pHi) of 0.14 +/- 0.02 pH units (n=6), which was not sensitive to ROCK inhibition. Therefore, dh-MotC activates Na+/H+ exchange via a ROCK-mediated cascade, and increases steady-state pHj in a ROCK-independent manner. Dh-MotC has been described as a Rho activator and is known to induce stress fibre and focal adhesion formation in serum-starved Swiss 3T3 cells. Inhibition of Na+/H+ exchange by HOE694 did not prevent dh-MotC-induced formation of stress fibres or focal adhesions. Similarly, the dh-MotC-mediated inhibition of tumour cell migration was not prevented by co-treatment with HOE694. These data suggest that increased Na+/H+ exchange activity is not required for dh-MotC induced stress fibres/focal adhesions and that dh-MotC inhibits tumour cell migration via a pathway that is independent of Na+/H+ exchange. Concentrations of dh-MotC that are effective at preventing tumour cell migration were toxic to normal mammary epithelial cells cultured on three dimensional basement membrane gels as evidenced by increased levels of active caspase-3 staining in dh-MotC treated mammary spheroids. Adherens and tight junction architecture was also disrupted under these conditions as evidenced by the loss of junctional E-cadherin and apically polarized ZO-1, components of adherens and tight junctions, respectively. In two-dimensional monolayer mammary epithelial cell cultures, dh-MotC did not affect cell morphology or localization of Ecadherin and ZO-1. Thus, there appears to be a differential response to dh-MotC between monolayer and spheroid cell culture.
Item Metadata
Title |
Dihydromotuporamine C increases Na⁺/H⁺exchange activity via a Rho-kinase-dependent pathway and induces disruption of intercellular junctions in mammary epithelia
|
Creator | |
Publisher |
University of British Columbia
|
Date Issued |
2003
|
Description |
Invasion and angiogenesis are two major steps in the progression towards
metastatic cancer. Dihydromotuporamine C (dh-MotC) inhibits both of these
processes. To better characterize the mechanism of dh-MotC action, I examined
the compound's effects on Na+/H+ exchange (NHE) activity and normal
mammary epithelial cell viability and junctional architecture.
In contrast to squalamine, an anti-angiogenic compound that inhibits Na+/H+
exchanger isoform 3 (NHE3), dh-MotC (5 or 10μM) increased the rate of
recovery from an imposed acid load in nominally bicarbonate free conditions.
This increased rate of recovery was inhibited by HOE694, a selective NHE
inhibitor, and the Rho-kinase (ROCK) inhibitor Y-27632. Dh-MotC also induced
an upward shift in steady state intracellular pH (pHi) of 0.14 +/- 0.02 pH units
(n=6), which was not sensitive to ROCK inhibition. Therefore, dh-MotC activates
Na+/H+ exchange via a ROCK-mediated cascade, and increases steady-state pHj
in a ROCK-independent manner.
Dh-MotC has been described as a Rho activator and is known to induce stress
fibre and focal adhesion formation in serum-starved Swiss 3T3 cells. Inhibition of
Na+/H+ exchange by HOE694 did not prevent dh-MotC-induced formation of
stress fibres or focal adhesions. Similarly, the dh-MotC-mediated inhibition of
tumour cell migration was not prevented by co-treatment with HOE694. These
data suggest that increased Na+/H+ exchange activity is not required for dh-MotC
induced stress fibres/focal adhesions and that dh-MotC inhibits tumour cell
migration via a pathway that is independent of Na+/H+ exchange.
Concentrations of dh-MotC that are effective at preventing tumour cell migration
were toxic to normal mammary epithelial cells cultured on three dimensional
basement membrane gels as evidenced by increased levels of active caspase-3
staining in dh-MotC treated mammary spheroids. Adherens and tight junction
architecture was also disrupted under these conditions as evidenced by the loss
of junctional E-cadherin and apically polarized ZO-1, components of adherens
and tight junctions, respectively. In two-dimensional monolayer mammary
epithelial cell cultures, dh-MotC did not affect cell morphology or localization of Ecadherin
and ZO-1. Thus, there appears to be a differential response to dh-MotC
between monolayer and spheroid cell culture.
|
Extent |
4067962 bytes
|
Genre | |
Type | |
File Format |
application/pdf
|
Language |
eng
|
Date Available |
2009-10-28
|
Provider |
Vancouver : University of British Columbia Library
|
Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
|
DOI |
10.14288/1.0091028
|
URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
|
Graduation Date |
2003-11
|
Campus | |
Scholarly Level |
Graduate
|
Aggregated Source Repository |
DSpace
|
Item Media
Item Citations and Data
Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.