| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Tumor Biology |
Fraser Laboratories, McGill University and Royal Victoria Hospital [M. T., K. S., D. L., C. B. S.], and Pharmaceutical Sector, Biotechnology Research Institute, National Research Council of Canada [S-H. S.], Montreal, Quebec, H3A 1A1 Canada
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A decrease in pHi could arise as a consequence of disrupted regulation of proton extrusion pathways that include BAF-1-sensitive H+-ATPase and amiloride inhibitable Na+/H+ antiport (NHE), which are maximally active at pHi of <6.7, and a Zn2+-inhibitable H+ conductance that is insensitive to BAF-1 and amiloride (5, 6, 7, 8, 9)
. Additional pH regulatory mechanisms involving sodium-dependent Cl-/HCO (3-) exchanger that functions optimally at pHi 
6.9 have also been described (10)
. Although modulation of pH-regulatory pathways by SHP-1 has not been shown, there is evidence that NHE and H+-ATPase could be activated in the presence of phosphotyrosine phosphatase inhibitors (11, 12, 13, 14)
. To define the relationship between SHP-1 and pHi, we investigated: (a) the requirement of a change in pHi for SST-induced translocation of SHP-1 to the membrane in MCF-7 cells; (b) the effect of (direct) acidification on cellular distribution of SHP-1 and apoptosis; (c) the ability of selective inhibitors of distinct proton extrusion pathways to induce intracellular acidification and apoptosis; and (d) the effect of acidification in two other breast cancer cell lines that displayed cytotoxic response to SST (T47D) or not (MDA-MB-231). We report here that SST-induced intracellular acidification occurs distal to or independent of changes in SHP-1, p53, and Bax and may arise due to inhibition of Na+/H+ exchanger and H+-ATPase. Additionally, SHP-1 is necessary not only for agonist-induced acidification but also for the execution of acidification-dependent apoptosis.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cell Culture.
MCF-7 cells were plated in 75-cm2 culture flasks and grown in MEM containing nonessential amino acids and supplemented with 10% FBS and 10 mg/ml bovine insulin. MCF-7 cells stably expressing SHP-1 or its catalytically inactive mutant SHP-1C455S were established as described previously and maintained in the above medium containing 400 µg/ml G-418 (2)
. T47D cells were maintained in RPMI 1640 supplemented with 10% FBS, and MDA-MB-231 cells were cultured in Liebovitz L-15 medium containing 10% FBS. Inhibition of individual proton extrusion pathways in MCF-7 cells was monitored using 2.5 µM Zn2+ (inhibitor of BAF-1 and amiloride-insensitive proton transport), 250 µM DIDS (inhibitor of Cl-/HCO3- channel), 100 nM BAF-1 (inhibitor of H+-ATPase), and 100 nM EIPA (inhibitor of NHE). When investigating the effect of intracellular acidification, the cells were cultured in the presence of the proton ionophore nigericin (10 µM) in medium buffered to the required pH ranging from 7.25 to 5.0. Nigericin-containing media were supplemented with 140 mM K+, which facilitates pH equilibration (15
, 16)
.
Measurement of PTP Activity SHP-1 Immunoblot Analysis.
To assess the effect of SST on translocation of SHP-1, flasks containing an equal number (5 x 106) of cells were incubated for 1 h in the absence or presence of 100 nM [D-Trp8]SST-14. Cells were washed in PBS and resuspended in buffer containing 200 mM mannitol, 68 mM sucrose, 50 mM HEPES-KOH (pH 7.4), 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM DTT, and protease inhibitors. After incubation for 30 min on ice, the cells were homogenized by hand in a Dounce homogenizer with a glass pestle. Homogenates were centrifuged at 14,000 x g for 20 min, and supernatants were kept at -80°C. Phosphatase activity in whole-cell extracts or membrane and cytosolic fractions was determined using pNPP as the substrate (2)
. Immunoblot analysis was carried out using rabbit polyclonal anti-SHP-1 antibody as described previously (4)
.
Detection of Apoptosis by Annexin V Labeling and DNA Fragmentation Analysis.
After treatment with the peptide, cells were incubated with FITC conjugated annexin V and the DNA intercalating dye propidium iodide using the apoptosis detection kit and analyzed by dual label flow cytometry according to the manufacturer’s instructions. To assess DNA fragmentation, cellular DNA was extracted twice with phenol/chloroform and once with chloroform from cells incubated in lysis buffer [500 mM Tris-HCl (pH 9) containing 2 mM EDTA, 10 mM NaCl, 1% SDS, and 1 mg/ml proteinase K] at 48°C for 30 h. DNA extracts were incubated with 300 mg/ml bovine pancreatic RNase A at 37°C for 1 h, and 10-mg aliquots of DNA samples containing 10 mg/ml ethidium bromide were subjected to inversion field gel electrophoresis on 1.2% (w/v) agarose gels using the Hoefer Switchback pulse controller and visualized under UV light.
Flow Cytometry.
Flow cytometry was carried out in a Becton Dickinson FACStar Vantage Plus flow cytometer. p53 and Bax were labeled in ethanol-fixed, permeabilized cells with wild-type specific anti-p53 antibody (pAb 1801; Oncogene) and anti-Bax antibody (C-21; Santa Cruz Biotechnology), followed by FITC-conjugated secondary antibody. FITC fluorescence in the cells was excited by a 5 W argon laser generating light at 351–363 nm and detected with a 560-nm short pass dichroic filter. At least 10,000 gated events were recorded for each sample, and the data were analyzed by Winlist software (Verity Software House, Topsham, ME).
Measurement of Intracellular pH.
Cells were incubated at 37°C in the absence or presence of 100 nM [D-Trp8]SST-14 for 24 h and loaded with 10 µM acetoxymethylester derivative of SNARF-1 during the final hour (2
, 17)
. The cells were then scraped, washed, and maintained at 37°C in a Becton Dickinson FACStar Vantage cytometer. Intracellular carboxy SNARF-1 was excited at 488 nm, and emission was recorded at both 580 and 640 nm with 5-nm band pass filters with linear amplifiers. The ratio of the emissions at these wavelengths was electronically calculated and used as a parameter indicative of intracellular pH. The intracellular pH values in control and treated cells were estimated by comparison of the mean ratios of the samples to a calibration curve of intracellular pH generated by incubation of carboxy-SNARF-1 loaded cells in nigericin containing buffers with adjusted pH ranging from 8.0 to 6.25 (2)
. Cells with fluorescence of <50 units were excluded in the calculation of the ratio of the emissions at 580 and 640 nm.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Addition of the proton ionophore nigericin to the incubation medium abolished the pH lowering effect of [D-Trp8]SST-14 (Fig. 1A)
as well as its ability to induce apoptosis (Fig. 1, B and C)
. Prevention of intracellular acidification by nigericin did not affect [D-Trp8]SST-14-induced increase in membrane-associated tyrosine phosphatase activity or the translocation of SHP-1 to the membrane (Fig. 2)
. Likewise, the inductive effect of [D-Trp8]SST-14 on wild-type p53 and Bax was also not dependent on pHi (Fig. 3)
. [D-Trp8]SST-14 induced increase in p53 was 8.0 ± 1.2 and 6.1 ± 0.56 fold, respectively, in the presence and absence of nigericin. The [D-Trp8]SST-14-induced increase in Bax (4.1 ± 0.76 and 3.2 ± 0.45 fold, respectively) was also comparable under these conditions.
|
|
|
. No apoptosis was evident in cells with a pHi >7.0 or in cells with pHi of <6.0. To our surprise, acidification-induced apoptosis in MCF-7 cells was abolished by the tyrosine phosphase inhibitor orthovanadate (Fig. 4B)
. We therefore assessed the phosphatase activity and the distribution of SHP-1 in the cytosolic and membrane fractions as a function of pHi. An increase in membrane-associated tyrosine phosphatase activity was seen in cells with pHi of 6.5 and 6.0 (Fig. 5A)
. A pH-dependent redistribution of SHP-1 was observed in cells with pHi of 6.5 and 6.0 but not in cells with more acidic or alkaline pHi (Fig. 5B)
. To confirm the SHP-1 dependency of acidification-induced cytotoxic signaling, we compared the effect of lowering the pHi to 6.5 in cells overexpressing the wild-type enzyme or its catalytically inactive mutant, SHP-1C455S. As shown in Fig. 6
, acidification-induced DNA fragmentation was enhanced by the overexpression of SHP-1 and was completely abolished by SHP-1C455S.
|
|
|
. The cellular pH did not decrease in response to Zn2+. The Cl-/HCO3- channel inhibitor DIDS caused mild acidification (pHi = 6.87 ± 0.07 compared with 7.24 ± 0.06 in the control; Fig. 7A
). By contrast, the H+-ATPase inhibitor BAF-1 and the NHE inhibitor EIPA lowered the intracellular pH by 0.60 and 0.69 units (pHi = 6.65 ± 0.06 and 6.55 ± 0.053), respectively. BAF-1 and EIPA together decreased the pHi to almost the same level as did [D-Trp8]SST-14 (pHi = 6.48 ± 0.04 and 6.42 ± 0.05, respectively). Apoptosis was seen to occur in cells treated with BAF-1 and EIPA but not with Zn2+ and DIDS (Fig. 7B)
. DNA fragmentation was greater in cells treated with EIPA than with BAF-1. The degree of apoptosis in cells treated simultaneously with BAF-1 and EIPA was comparable with that induced by [D-Trp8]SST-14. To determine which of these channels are modulated by SHP-1, we compared the effect of DIDS, BAF-1, and EIPA in MCF-7 cells overexpressing the wild-type SHP-1 or its catalytically inactive mutant. The ability of DIDS to decrease the pHi was not affected by the overexpression of either SHP-1 or SHP-1C455S (Fig. 8)
. By contrast, BAF-1 and EIPA, which were fully capable of triggering intracellular acidification in cells overexpressing SHP-1, were ineffective in acidifying SHP-1C455S-expressing cells.
|
|
. By contrast, both cells underwent apoptosis when incubated in media buffered to pH 6.5 in the presence of nigericin. Furthermore, acidification-induced apoptosis was inhibited by orthovanadate in both cell types. Additionally, [D-Trp8]SST-14 induced translocation of SHP-1 to the membrane in T47D but not in MDA-MB 231 cells, whereas there was an increase in membrane-associated SHP-1 in both cells in which the pHi was clamped to 6.5 (Fig. 9, B and C)
. [D-Trp8]SST-14-induced apoptosis in T47D cells was associated with intracellular acidification (pHi = 6.46 ± 0.06) comparable with that seen in MCF-7 cells. Interestingly, despite its inability to signal apoptosis in MDA-MB-231 cells, [D-Trp8]SST-14 induced a slight, but significant, decrease in pHi (6.95 ± 0.07 versus 7.3 ± 0.07; Fig. 10
).
|
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The inability of DIDS to trigger apoptosis, although it decreased the pHi to 6.9, suggests that inhibition of Na+-dependent Cl-/HCO3- channel is insufficient to trigger the apoptotic process. [D-Trp8]SST-14 decreased the pHi of MDA-MB-231 cells to 6.95, the same extent as did DIDS. The modest decrease in pHi induced by DIDS was comparable in MCF-7 cells expressing the wild-type enzyme or the inactive mutant (data not shown), suggesting that SST may inhibit Na+-dependent Cl-/HCO3- channel in an SHP-1-independent manner.
NHE and H+-ATPase are present in tumor cells, and their inhibition leads to intracellular acidification and apoptosis (18, 19, 20, 21, 22) . The present data showing that BAF-1 and EIPA trigger acidification and apoptosis in MCF-7 cells is in agreement with these findings. We showed that apoptosis resulting from the inhibition of NHE by EIPA was greater than that due to inhibition of H+-ATPase by BAF-1. The degree of acidification induced by EIPA was greater than that caused by BAF-1, suggesting that SST may exert a greater inhibitory effect on NHE compared with H+-ATPase. Alternatively, this may reflect the possibility that NHE is more active in pH regulation compared with H+-ATPase. The extent of acidification and apoptosis induced by these inhibitors together was comparable with that seen in [D-Trp8]SST-14-treated cells. Thus, inhibition of both H+-extrusion pathways may be required for the distal events of SST-induced cytotoxic signaling. The present finding that SHP-1C455S prevented the acidification caused by EIPA and BAF-1 suggests that the activity of both NHE and H+-ATPase is modulated by SHP-1. Interestingly, the resting pHi of MCF-7 cells was decreased by overexpressed SHP-1, whereas it was increased by SHP-1C455S (7.06 ± 0.08 and 7.37 ± 0.06, respectively, compared with the value of 7.25 + 0.07 in untransfected cells). These data, to our knowledge, provide the first documentation of SHP-1-dependent regulation of pH homeostasis through NHE and H+-ATPase. Although the specific involvement of SHP-1 in intracellular acidification has not been described previously, PTP inhibitors have been shown to induce multiple pH regulatory pathways (H+-ATPase, NHE, and a Zn2+-sensitive H+ conductance; Ref. 11 ). Vanadate was also capable of activating NHE in a variety of systems (12, 13, 14) . The activity of NHE and H+-ATPase has been shown to be modulated by serine/threonine, but not tyrosine, phosphorylation (23) . BAF-1 and EIPA failed to trigger acidification in cells expressing SHP-1C455S to the same extent as they did in MCF-7 cells (data not shown). We do not know at the present time how SST inhibits the activity of these proteins in an SHP-1-dependent manner. It remains to be established as to whether SHP-1 regulates the activity of NHE and H+-ATPase directly or indirectly through other proteins. Six isoforms of NHE have been identified (23) . NHE-1 and NHE-6 are ubiquitously expressed in most cell types. NHE-1 is found on the plasma membranes of virtually all cells and is believed to control cytosolic pH and cell volume regulation. NHE-2, NHE-3, NHE-4, and NHE-5 are more restricted in their expression patterns, reflecting specialized functions, and display differences in their apparent H+ sensitivity and relative, but not absolute, selectivity toward different inhibitors (23 , 24) . Such differences may arise due to a possible intracellular rather than plasma membrane localization of some NHEs. Indeed, NHE-3 has been shown to be predominantly juxtanuclear or endosomal (25) . The expression patterns and subcellular localization of the NHE isoforms in MCF-7 cells remain to be determined; hence, we do not know at the present time which specific NHE isoform(s) is inhibited by SST.
Cytotoxic signaling by SST is dependent not only on the recruitment of SHP-1 to the membrane but also on intracellular acidification. Such an effect seen in breast cancer cells is signaled in a subtype-selective manner uniquely via hSSTR subtype 3 in heterologous expression system in CHO-K1 cells, whereas the other four hSSTR subtypes elicit cytostatic response, resulting in G1 cell cycle arrest but not apoptosis (26 , 27) . The presence of hSSTR3 has been detected by reverse transcription-PCR in many, but not all, cancers of the breast and other tissues (28, 29, 30) . In tumors that do not express this subtype, SST analogues will fail to trigger apoptosis and may, therefore, have limited therapeutic potential. We have shown that intracellular acidification is sufficient to trigger apoptosis in an SHP-1-dependent manner, even in cells that do not undergo apoptosis in response to SST. The pHi of tumor cells is generally higher than that of the extracellular environment (31) . Indeed, compounds that reverse the imbalance between the intra- and extracellular pH of tumor cells in vivo have been developed as a potential new class of anticancer drugs (32 , 33) . Specifically, inhibitors of proton extrusion have been shown to inhibit tumor cell growth (34, 35, 36, 37, 38) . The importance of tyrosine phosphatases in antiproliferative signaling has been recognized but has not yet been exploited for their therapeutic potential. Our finding that SHP-1 is involved in both receptor-mediated and acidification-induced apoptotic signaling underscores its potential usefulness in cancer therapy.
In summary, we have shown that SST-induced acidification appears to be due to SHP-1-dependent inhibition of NHE and H+-ATPase and SHP-1-independent inhibition of Na+-dependent Cl-/HCO3- channel. SST-induced acidification occurs independent of or distal to the induction of wild-type p53 and Bax. SHP-1 is necessary not only for agonist-induced acidification but also for apoptosis triggered by a decrease in pHi. A pHi-dependent membrane association and activation of SHP-1 occurs in breast cancer cell lines that undergo apoptosis in response to SST (MCF-7 and T47D) or not (MDA-MB 231). These findings reveal a hitherto unrecognized interdependency between intracellular acidification and SHP-1-dependent signaling of apoptosis. We suggest that combined targeting of SHP-1 and intracellular acidification may lead to a novel strategy of anticancer therapy, bypassing the need for receptor-mediated signaling.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was supported by a grant from the Canadian M.R.C. (to C. B. S.). K. S. is a recipient of a studentship from the Fonds de la Recherche en Sante du Quebec. M. T. is a recipient of Royal Victoria Hospital Research Institute fellowship. 
2 To whom requests for reprints should be addressed, at Fraser Laboratories, M3.15, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, H3A 1A1 Canada. Phone: (514) 842-1231, extension 5359; Fax: (514) 849-3681; E-mail: mdcs{at}musica.mcgill.ca
3 The abbreviations used are: SST, somatostatin; SSTR, somatostatin receptor; hSSTR, human SSTR; pHi, intracellular pH; BAF-1, bafilomycin A-1; DIDS, 4,4'-diisothicyanatostilbene-2,2'-disulfonic acid; EIPA, 5-(N-ethyl-N-isopropyl)-amiloride; NHE, Na+/H+ exchanger; pNPP, p-nitrophenyl phosphate; FBS, fetal bovine serum.
4 K. Sharma, M. Thangaraju, S-H. Shen, and C. B. Srikant, submitted for publication.
Received 10/27/98. Accepted 2/ 5/99.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. HARGUINDEY, J. L. ARRANZ, M. L. WAHL, G. ORIVE, and S. J. RESHKIN Proton Transport Inhibitors as Potentially Selective Anticancer Drugs Anticancer Res, June 1, 2009; 29(6): 2127 - 2136. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Xue, D. Zhou, H. Yao, O. Gavrialov, M. J. McConnell, B. D. Gelb, and G. G. Haddad Novel functional interaction between Na+/H+ exchanger 1 and tyrosine phosphatase SHP-2 Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2007; 292(6): R2406 - R2416. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. F. Pedersen, M. E. O'Donnell, S. E. Anderson, and P. M. Cala Physiology and pathophysiology of Na+/H+ exchange and Na+-K+-2Cl- cotransport in the heart, brain, and blood Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2006; 291(1): R1 - R25. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Kokkonen, A. Rivinoja, A. Kauppila, M. Suokas, I. Kellokumpu, and S. Kellokumpu Defective Acidification of Intracellular Organelles Results in Aberrant Secretion of Cathepsin D in Cancer Cells J. Biol. Chem., September 17, 2004; 279(38): 39982 - 39988. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Reshkin, A. Bellizzi, R. A. Cardone, M. Tommasino, V. Casavola, and A. Paradiso Paclitaxel Induces Apoptosis via Protein Kinase A- and p38 Mitogen-activated Protein-dependent Inhibition of the Na+/H+ Exchanger (NHE) NHE Isoform 1 in Human Breast Cancer Cells Clin. Cancer Res., June 1, 2003; 9(6): 2366 - 2373. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Pang, S. Wakabayashi, and M. Shigekawa Expression of Calcineurin B Homologous Protein 2 Protects Serum Deprivation-induced Cell Death by Serum-independent Activation of Na+/H+ Exchanger J. Biol. Chem., November 8, 2002; 277(46): 43771 - 43777. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Yusta, J. Estall, and D. J. Drucker Glucagon-like Peptide-2 Receptor Activation Engages Bad and Glycogen Synthase Kinase-3 in a Protein Kinase A-dependent Manner and Prevents Apoptosis following Inhibition of Phosphatidylinositol 3-Kinase J. Biol. Chem., July 5, 2002; 277(28): 24896 - 24906. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-C. Hsu, L. D. Shultz, X. Su, J. Shi, P.-A. Yang, M. J. Relyea, H.-G. Zhang, and J. D. Mountz Mutation of the Hematopoietic Cell Phosphatase (Hcph) Gene Is Associated with Resistance to {{gamma}}-Irradiation-Induced Apoptosis in Src Homology Protein Tyrosine Phosphatase (SHP)-1-Deficient ""Motheaten"" Mutant Mice J. Immunol., January 15, 2001; 166(2): 772 - 780. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. P. Boushey, B. Yusta, and D. J. Drucker Glucagon-like Peptide (GLP)-2 Reduces Chemotherapy-associated Mortality and Enhances Cell Survival in Cells Expressing a Transfected GLP-2 Receptor Cancer Res., January 1, 2001; 61(2): 687 - 693. [Abstract] [Full Text]
|
|
|
|
|
|
D. Liu, G. Martino, M. Thangaraju, M. Sharma, F. Halwani, S.-H. Shen, Y. C. Patel, and C. B. Srikant Caspase-8-mediated Intracellular Acidification Precedes Mitochondrial Dysfunction in Somatostatin-induced Apoptosis J. Biol. Chem., March 24, 2000; 275(13): 9244 - 9250. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Scott and N. S. Allen Changes in Cytosolic pH within Arabidopsis Root Columella Cells Play a Key Role in the Early Signaling Pathway for Root Gravitropism Plant Physiology, December 1, 1999; 121(4): 1291 - 1298. [Abstract] [Full Text]
|
|
|
|
|
|
M. Thangaraju, K. Sharma, B. Leber, D. W. Andrews, S.-H. Shen, and C. B. Srikant Regulation of Acidification and Apoptosis by SHP-1 and Bcl-2 J. Biol. Chem., October 8, 1999; 274(41): 29549 - 29557. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Yusta, R. P. Boushey, and D. J. Drucker The Glucagon-like Peptide-2 Receptor Mediates Direct Inhibition of Cellular Apoptosis via a cAMP-dependent Protein Kinase-independent Pathway J. Biol. Chem., November 3, 2000; 275(45): 35345 - 35352. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
