Cancer Research Meeting Calendar Frontiers in Basic Cancer Research
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
Cancer Research 67, 3337, April 1, 2007. doi: 10.1158/0008-5472.CAN-06-2480
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, X.-F.
Right arrow Articles by Neuzil, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, X.-F.
Right arrow Articles by Neuzil, J.

Experimental Therapeutics, Molecular Targets, and Chemical Biology

A Peptide Conjugate of Vitamin E Succinate Targets Breast Cancer Cells with High ErbB2 Expression

Xiu-Fang Wang1, Marc Birringer3, Lan-Feng Dong1, Pavel Veprek4, Pauline Low2, Emma Swettenham1, Marina Stantic1, Lin-Hong Yuan6, Renata Zobalova1,5, Kun Wu6, Miroslav Ledvina4, Stephen J. Ralph2 and Jiri Neuzil1,5

1 Apoptosis Research Group and 2 Genomics Research Centre, School of Medical Science, Griffith University, Southport, Queensland, Australia; 3 Department of Human Nutrition, Institute of Nutrition, Friedrich-Schiller University, Jena, Germany; 4 Institute of Organic Chemistry and Biochemistry and 5 Molecular Therapy Group, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic; and 6 Department of Food and Nutrition, Harbin Medical University, Harbin, Heilongjiang Province, China

Requests for reprints: Jiri Neuzil, Apoptosis Research Group, School of Medical Science, Griffith Institute of Health and Medical Research, Griffith University Gold Coast Campus, Southport, Queensland, Australia. Phone: 61-7-55529109; Fax: 61-7-55524888; E-mail: j.neuzil{at}griffith.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Overexpression of erbB2 is associated with resistance to apoptosis. We explored whether high level of erbB2 expression by cancer cells allows their targeting using an erbB2-binding peptide (LTVSPWY) attached to the proapoptotic {alpha}-tocopheryl succinate ({alpha}-TOS). Treating erbB2-low or erbB2-high cells with {alpha}-TOS induced similar levels of apoptosis, whereas {alpha}-TOS-LTVSPWY induced greater levels of apoptosis in erbB2-high cells. {alpha}-TOS rapidly accumulated in erbB2-high cells exposed to {alpha}-TOS-LTVSPWY. The extent of apoptosis induced in erbB2-high cells by {alpha}-TOS-LTVSPWY was suppressed by erbB2 RNA interference as well as by inhibition of either endocytotic or lysosomal function. {alpha}-TOS-LTVSPWY reduced erbB2-high breast carcinomas in FVB/N c-neu transgenic mice. We conclude that a conjugate of a peptide targeting {alpha}-TOS to erbB2-overexpressing cancer cells induces rapid apoptosis and efficiently suppresses erbB2-positive breast tumors. [Cancer Res 2007;67(7):3337–44]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Receptor tyrosine kinases, such as erbB2/HER2 (a product of the c-neu gene), render cancer cells resistant to apoptosis treatment and make patients with erbB2-positive carcinomas a therapeutic challenge (1, 2). The major problem associated with high expression of erbB2 is autophosphorylation of the receptor and the ensuing activation of growth signaling pathways and proangiogenic and antiapoptotic mechanisms.

ErbB2 is overexpressed in ~30% of breast cancers. A major complication associated with its high expression is linked to activation of Akt (3, 4), a serine/threonine kinase that promotes cellular survival (5) due to phosphorylation of proteins like caspase-9 (6), Bad (7), or Bax (8). Akt also causes activation of nuclear factor-{kappa}B (9) that controls expression of prosurvival genes, such as the inhibitor of apoptosis proteins family members (10, 11) and the caspase-8 inhibitor FLIP (12).

We have studied the molecular mechanism of the proapoptotic and antineoplastic activity of vitamin E analogues, epitomized by the redox-silent {alpha}-tocopheryl succinate ({alpha}-TOS; refs. 13, 14). This agent has been proven efficient in selective induction of apoptosis in cancer cells (15) and in suppression of experimental cancer (16–20). We showed that {alpha}-TOS can bypass mutations or deficiencies in important tumor suppressor genes such as p53 or p21Waf1/Cip1 (18). Recently, our results documented that {alpha}-TOS can also kill breast cancer cells overexpressing erbB2 (21).

A limiting point in efficient delivery of anticancer drugs is their uptake by malignant cells. Uptake of {alpha}-TOS is relatively slow because it, most likely, relies on passive diffusion. This follows from experiments, in which acidification of media enhanced uptake by cancer cells of {alpha}-TOS, a weak acid, because its free COOH group is protonated to a higher extent at lower pH (22). We decided to explore whether targeting {alpha}-TOS to erbB2-overexpressing cells by generating a vitamin E analogue conjugated to a specific peptide provides a method for its efficient delivery to breast cancer cells resistant to conventional therapy. We based our hypothesis on the report by Shadidi and Sioud (23), who identified the heptapeptide LTVSPWY to target erbB2 antisense oligodeoxynucleotides to breast cancer cells, suppressing expression of the c-neu gene. We show here that the peptide {alpha}-TOS-LTVSPWY conjugate efficiently kills breast cancer cells with high levels of erbB2. Our data suggest that peptides binding to receptors expressed on cancer cells coupled to inducers of apoptosis may efficiently target cancer cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. ErbB2-low MCF-7 and erbB2-high MDA-MB-453 cells as well as the MCF-7/HER2-18 cells stably transfected with an erbB2 vector (24, 25) were cultured in DMEM supplemented with 10% FCS and antibiotics. To inhibit lysosomal function, culture medium was supplemented with 20 mmol/L NH4Cl. In some cases, cells were cotreated with 25 µmol/L each of the pan-caspase, caspase-8, or caspase-9 inhibitors Z-VAD-FMK, Z-IETD-FMK, or Z-LEHD-FMK, respectively (Calbiochem, La Jolla, CA).

Synthesis of {alpha}-TOS-peptide conjugate. The H-Tyr(t-Bu)-Trp(t-Boc)-Pro-Ser(t-Bu)-Val-Thr(t-Bu)-Leu-Rink amide was prepared according to the standard Fmoc protocol on a LIPS Vario Peptide synthesizer. All acylation reactions were carried out for 1 h using a 10-fold excess of Fmoc-amino acids activated with TBTU (1 equivalent) in the presence of DIPEA (2 equivalents) and HOBt (1 equivalent). The NH2-terminal conjugation was carried out by activation of {alpha}-TOS with 1 equivalent of PyBOP in the presence of HOBt (1 equivalent) and DIPEA (2 equivalents). The conjugated peptide was cleaved from the resin using trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5) for 2 h. The highly lipophilic peptide conjugate was extracted with diethylether and validated by analysis with high-performance liquid chromatography (HPLC) and electrospray-mass spectrometry (expected MH+, 1378.8; found MH+, 1378.7).

Assessment of apoptosis, reactive oxygen species formation, and mitochondrial potential. Apoptosis was quantified by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA) using the Annexin V method as described elsewhere (26). Cellular reactive oxygen species (ROS) were assessed by flow cytometry using the fluorescent probe dihydroethidium (Molecular Probes, Eugene, OR; ref. 27). Relative levels of the ROS signal were expressed as a change in mean fluorescence intensity. The mitochondrial inner trans-membrane potential ({Delta}{Psi}m) was assessed using the polychromatic dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidoazolyl-carbocyanino iodide (JC-1) as detailed elsewhere (26), and the population of cells with increased green fluorescence was estimated by flow cytometry. In some experiments, cells were pretreated for 2 h with 2.5 µmol/L mitochondrially targeted coenzyme Q (MitoQ), a triphenylphosphonium adduct of coenzyme Q that accumulates in the mitochondrial inner membrane and acts as an efficient scavenger of mitochondrial ROS (28).

Western blotting. Cells were lysed and stored at –80°C until analysis. Protein levels were quantified using the bicinchoninic assay. Protein samples (80 µg per lane) were denatured, resolved using 12.5% SDS-PAGE, and transferred onto a nitrocellulose membrane. The membrane was blocked and incubated overnight with anti-dynamin-2 or anti-erbB2 IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Anti-ß-actin IgG (Santa Cruz Biotechnology) was used as a loading control. After incubation with a horseradish peroxidase–conjugated secondary IgG (Amersham, Arlington Heights, IL), the blots were developed using the ECL kit (Pierce, Rockford, IL).

Cell transfection. The plasmids used were Bcl-xL-EGFP (29) and wild-type (WT) and dominant-negative (DN; K44A) dynamin-2 (30). Transfections were carried out as described previously (26). In brief, MDA-MB-453 cells at 50% to 70% confluence were supplemented with 5 µg DNA preincubated for 10 min with 10 µL LipofectAMINE 2000 (Life Technologies, Gaithersburg, MD) and 1 mL Opti-MEM. After 3 to 4 h, cells were washed and supplemented with complete DMEM and left for 24 h, at which stage the cells were incubated with complete DMEM supplemented with G418. The cells were maintained in the selection medium for at least five passages and assessed for protein expression by fluorescence microscopy (Bcl-xL) or Western blotting (dynamin-2).

RNA interference. ErbB2-specific and validated short interfering RNA (siRNA) oligonucleotides and nonspecific scrambled siRNA were purchased from Ambion (Austin, TX). Transfection of MDA-MB-453 cells with siRNA was done as reported elsewhere (27). Briefly, cells were allowed to reach ~50% confluency and supplemented with 60 pmol/L siRNA preincubated with Oligofectamine (Invitrogen, San Diego, CA) and overlayed with Opti-MEM. Cells were washed 24 h later with PBS, overlayed with complete DMEM, and cultured for additional 24 h, and transgene expression was confirmed before using the cells for further experiments.

Endocytosis assay. Low-density lipoprotein (LDL) uptake, mediated by endocytosis dependent on the LDL receptor, was assessed as reported (31). LDL was labeled with the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) for 8 h at 37°C. MDA-MB-453 parental cells, and the WT and DN dynamin-2 transfectants at ~50% confluency were incubated with DiI-labeled LDL for 2 h at 37°C, and the level of internalized DiI-LDL estimated by flow cytometry. Endocytosis was also assessed using FITC-conjugated transferrin (FITC-Tr). In brief, FITC-Tr (Molecular Probes) at 0.1 mg protein per mL was added to cells at ~50% confluency; the cells were incubated in serum-free DMEM for 1 h at 37°C and assessed by flow cytometry.

Mouse tumor experiments. A colony of transgenic FVB/N202 c-neu mice carrying the rat HER-2/neu proto-oncogene driven by the mouse mammary tumor virus promoter on the H-2q FVB/N background (32) was established at the Griffith University Animal Facility and maintained under strict inbreeding conditions. The presence of the transgene was routinely confirmed by PCR. Some 70% of the female mice developed spontaneous mammary carcinomas with a mean latency time of >7 months. Female transgenic FVB/N c-neu mice bearing progressively growing tumors with mean volume of 25 mm3 were randomly assigned to control or treatment groups. Tumor size was quantified by ultrasound imaging using the Vevo770 instrument and the 40-MHz RMV704 scan-head (both Visualsonics, Toronto, Ontario, Canada) allowing 40-µm resolution of individual scans as well as by digital calipers. Mice received treatment with the excipient (corn oil/4% ethanol) alone (control), 15 or 5 µmol {alpha}-TOS, or 5 or 2.5 µmol {alpha}-TOS-LTVSPWY given by i.p. injection every 3 to 4 days.

Animal experiments were done according to the guidelines of the Australian and New Zealand Council for the Care and Use of Animals in Research and Teaching and were approved by the Griffith University Animal Ethics Committee.

{alpha}-TOS analysis. Intracellular levels of {alpha}-TOS were quantified using an HPLC method (33).

Statistics. Data were obtained from at least three different experiments, and the results are presented as mean ± SD. Significance between data points was assessed using the Student's t test, and data were considered significantly different when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We first tested whether conjugating {alpha}-TOS to a peptide recognized by erbB2 would target the vitamin E analogue to cells overexpressing the receptor. To do this, we prepared a conjugate of {alpha}-TOS with the heptapeptide LTVSPWY by its addition to the free carboxylate on the succinyl moiety of the vitamin E ester. We found that erbB2-low (MCF-7) and erbB2-high cells (MCF-7/HER2-18 and MDA-MB-453) were all similarly sensitive to {alpha}-TOS, whereas erbB2-low cells were relatively resistant to {alpha}-TOS-LTVSPWY (Fig. 1 ). However, the peptide conjugate of {alpha}-TOS was significantly more effective in inducing apoptosis in erbB2-high cells than was {alpha}-TOS (Fig. 1). The LTVSPWY peptide itself did not induce any detectable apoptosis (data not shown).


Figure 1
View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Induction of apoptosis in breast cancer cells exposed to {alpha}-TOS or {alpha}-TOS-LTVSPWY. MCF-7 (A and B), MDA-MB-453 (C and D), and MCF-7/HER2-18 cells (E and F) were incubated with {alpha}-TOS (A, C, and E) or {alpha}-TOS-LTVSPWY (B, D, and F) at 25 or 50 µmol/L for 6, 12, and 24 h and assessed for apoptosis using the Annexin V method. E, inset, level of expression of erbB2 in MCF-7, MDA-MB-453, and MCF-7/HER2-18 cells using Western blotting. Points, mean (n = 3); bars, SD. Representative Western blotting of three independent experiments. * and **, significantly different apoptosis data in erbB2-low and erbB2-high cells exposed to {alpha}-TOS or {alpha}-TOS-peptide at 25 and 50 µmol/L, respectively.

 
Previous reports showed that {alpha}-TOS induced apoptosis by the intrinsic pathway (26, 27, 34, 35). We tested whether mitochondria and the ensuing events are also involved in killing of MDA-MB-453 cells exposed to {alpha}-TOS-LTVSPWY. We first studied the effect of {alpha}-TOS-LTVSPWY on generation of ROS and dissipation of {Delta}{Psi}m as early indicators of apoptosis preceding the onset of the commitment phase. Figure 2A and B documents that the peptide conjugate caused a more rapid accumulation of ROS and {Delta}{Psi}m dissipation in MDA-MB-453 cells than did {alpha}-TOS. MitoQ is a potent antioxidant that is targeted to the mitochondrial inner membrane (27), and using this agent revealed that the effect of {alpha}-TOS-LTVSPWY on mitochondrial destabilization was caused by mitochondrially derived ROS because inhibition of ROS accumulation and dissipation of {Delta}{Psi}m by the addition of MitoQ also inhibited the peptide conjugate–induced apoptosis (data not shown). We next tested whether apoptosis induced by {alpha}-TOS-LTVSPWY was dependent on caspases. Figure 2C shows that the pan-caspase inhibitor and the caspase-9 inhibitor suppressed apoptosis induction in MDA-MB-453 cells exposed to both {alpha}-TOS-LTVSPWY and {alpha}-TOS, whereas this was not observed in case of the caspase-8 inhibitor. We also studied whether overexpression of the mitochondrial antiapoptotic protein Bcl-xL protects MDA-MB-453 cells from apoptosis induced by {alpha}-TOS-LTVSPWY. Figure 2D shows that apoptosis induced by {alpha}-TOS-LTVSPWY was inhibited in erbB2-overexpressing cells transfected with Bcl-xL. Collectively, these data confirm that mitochondria are important for apoptosis induction by the peptide conjugate in erbB2-overexpressing cells.


Figure 2
View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. {alpha}-TOS-LTVSPWY–induced apoptosis is mediated by the intrinsic pathway. MCF-7 and MDA-MB-453 cells were exposed to 50 µmol/L {alpha}-TOS or {alpha}-TOS-LTVSPWY for the periods (h) shown. The cells were evaluated for ROS generation using dihydroethidium (A) and for {Delta}{Psi}m using JC-1 (B). C, effect of the pan-caspase, caspase-8 (Casp8), or caspase-9 (Casp9) inhibitors (all 25 µmol/L) on apoptosis induced by 50 µmol/L {alpha}-TOS or {alpha}-TOS-LTVSPWY, as evaluated by the Annexin V assay. D, effect of overexpression of Bcl-xL on apoptosis induction by 50 µmol/L {alpha}-TOS or {alpha}-TOS-LTVSPWY in MDA-MB-453 cells. Columns, mean (n = 3); bars, SD. *, significantly different values obtained in MCF-7 and MDA-MB-453 cells (A and B). *, significantly different data from control values (C and D). MFI, mean fluorescence intensity.

 
To find out whether rapid killing of MDA-MB-453 cells by {alpha}-TOS-LTVSPWY was due to high levels of erbB2, we knocked down the receptor using erbB2 siRNA. Figure 3A shows that siRNA treatment for 48 h suppressed expression of erbB2. MDA-MB-453 cells pretreated with erbB2 siRNA were significantly more resistant to {alpha}-TOS-LTVSPWY but not to {alpha}-TOS alone (Fig. 3B).


Figure 3
View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Knockdown of erbB2 suppresses apoptosis in MDA-MB-453 cells exposed to {alpha}-TOS-LTVSPWY. MDA-MB-453 cells were treated with the vehicle (control cells), or erbB2-specific or scrambled siRNA. A, after 24 and 48 h, the cells were evaluated by Western blotting for expression of the erbB2 protein. B, the cells exposed to the erbB2 or scrambled siRNA for 48 h were treated with 50 µmol/L {alpha}-TOS or {alpha}-TOS-LTVSPWY for 12 and 24 h and assessed for apoptosis using the Annexin V method. Columns, mean (n = 3); bars, SD. Representative Western blotting images of three independent experiments. *, statistically different data for siRNA-treated versus nontreated cells.

 
A plausible explanation for the greater efficacy of apoptosis induced in erbB2-overexpressing cells by {alpha}-TOS-LTVSPWY is that the conjugate is rapidly endocytosed. To address this, we inhibited endocytosis of MDA-MB-453 cells by overexpression of DN dynamin-2 (30). Figure 4A reveals that the transfected cells expressed higher levels of either the WT or DN dynamin-2, and that the resulting inhibition of endocytosis suppressed apoptosis induced in MDA-MB-453 cell by the {alpha}-TOS-peptide conjugate, whereas levels of apoptosis triggered by {alpha}-TOS alone in either the control or the endocytosis-inhibited cells were not affected. Figure 4A (inset) documents that DN dynamin-2 inhibits endocytosis because overexpression of DN dynamin-2 inhibited the uptake of both LDL and transferrin.


Figure 4
View larger version (21K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Inhibition of endocytosis and lysosomal function suppresses {alpha}-TOS-LTVSPWY–induced apoptosis in MDA-MB-453 cells. A, MDA-MB-453 cells or their counterparts overexpressing WT or DN dynamin-2 were exposed to {alpha}-TOS or {alpha}-TOS-LTVSPWY at 50 µmol/L for various time periods and evaluated for apoptosis using the Annexin V method. Top inset, expression of dynamin-2 using Western blotting in parental cells (Par), or cells overexpressing WT or DN dynamin-2. Bottom inset, endocytosis of DiI-labeled LDL or FITC-Tr by parental MDA-MB-453 cells (column 1), or cells overexpressing WT (column 2) or DN dynamin-2 (column 3). Columns, mean fluorescence intensity. B, MDA-MB-453 cells were either left in the original medium or medium supplemented with 20 mmol/L NH4Cl. The cells were then exposed for various periods to {alpha}-TOS or {alpha}-TOS-LTVSPWY at 50 µmol/L and assessed for apoptosis using the Annexin V method. Columns, mean (n = 3); bars, SD. Representative Western blotting images of three independent experiments. *, statistically different data for cells transfected with DN dynamin-2 versus parental cells or versus cells transfected with WT dynamin-2.

 
An important feature of the peptide conjugate of {alpha}-TOS is that the bond between the heptapeptide and the vitamin E analogue can be hydrolyzed once inside cells, liberating the apoptogenic, free {alpha}-TOS. This follows from our previous work, in which we showed that masking the free carboxylate of vitamin E analogues by methylation negated their ability to kill cancer cells (36, 37). We tested whether lysosomal function was important for apoptosis induction by {alpha}-TOS-LTVSPWY due to presence of peptidases in the organelles. Addition of ammonium chloride to cells, which buffers the acidic pH of lysosomes, suppressed apoptosis induction in MDA-MB-453 cells by {alpha}-TOS-LTVSPWY, whereas apoptosis induced by {alpha}-TOS alone was not affected (Fig. 4B). This suggests involvement of lysosomes in conversion of the {alpha}-TOS-peptide conjugate into the proapoptotic {alpha}-TOS.

The above results support a role for overexpression of erbB2 in rapid uptake of {alpha}-TOS-LTVSPWY and in liberating the apoptogenic {alpha}-TOS. We therefore evaluated whether intracellular levels of free {alpha}-TOS in MCF-7 and MDA-MB-453 cells were altered after exposure to {alpha}-TOS-LTVSPWY or {alpha}-TOS. Figure 5 shows that MCF-7 cells accumulated free {alpha}-TOS more rapidly when exposed to the vitamin E analogue than when incubated with {alpha}-TOS-LTVSPWY. On the other hand, MDA-MB-453 cells accumulated free {alpha}-TOS fast when exposed to {alpha}-TOS-LTVSPWY, while showing slow {alpha}-TOS accumulation when exposed to the free vitamin E analogue. Very slow kinetics of {alpha}-TOS accumulation was observed when MDA-MB-453K44A cells were exposed to {alpha}-TOS-LTVSPWY (Fig. 5B), or when MDA-MB-453 cells were exposed to the peptide conjugate of the vitamin E analogue in the presence of ammonium chloride (Fig. 5C).


Figure 5
View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. {alpha}-TOS rapidly accumulates in MDA-MB-453 cells exposed to {alpha}-TOS-LTVSPWY. MCF-7 (A) and MDA-MB-453 or MDA-MB-453K44A (B) cells were exposed to 50 µmol/L {alpha}-TOS or {alpha}-TOS-LTVSPWY ({alpha}-TOS-P) for different periods and assessed for intracellular levels of {alpha}-TOS. C, effect of NH4Cl on {alpha}-TOS levels in MDA-MB-453 cells exposed to 50 µmol/L {alpha}-TOS or {alpha}-TOS-LTVSPWY for indicated periods. Points, mean (n = 3); bars, SD. *, statistically different data from cells exposed to {alpha}-TOS and {alpha}-TOS-peptide (A) and from cells exposed to {alpha}-TOS-peptide and {alpha}-TOS/{alpha}-TOS-peptide K44A (B). *, statistically different data obtained from cells exposed to {alpha}-TOS-peptide in the absence and presence of NH4Cl (C).

 
To determine the anticancer efficacy of the peptide conjugate of {alpha}-TOS, transgenic mice were used that spontaneously form ductal breast carcinomas due to overexpression of erbB2 in the mammary epithelial cells (32). Female mice that developed breast tumors were injected with {alpha}-TOS (15 or 5 µmol per mouse per dose) or {alpha}-TOS-LTVSPWY (5 or 2.5 µmol per mouse per dose), and the volume of the tumors was measured by ultrasound imaging, allowing precise quantification of tumors, as well as by calipers. Figure 6 shows that treatment with the peptide conjugate at 5 µmol reduced the initial volume of breast carcinomas by some 70% at a 3-fold lower dose than that of {alpha}-TOS, which reduced the tumors by about 50%. The effect of the {alpha}-TOS-peptide conjugate at 2.5 µmol was initially similar to that of {alpha}-TOS at 15 µmol but failed to reduce the tumor size, as observed also for {alpha}-TOS at 5 µmol. The effect of reduction in tumor size was apparent after one dose of the peptide-{alpha}-TOS conjugate at 5 µmol, whereas this was delayed when {alpha}-TOS at 15 µmol was used. We also compared the use of ultrasound imaging and calipers for monitoring tumor volume. The data shown in Fig. 6A and B clearly document the superiority of ultrasound imaging over calipers. In many cases, majority of the tumor is embedded in the body of the animal with as little as 10% of the tumor accessible for mechanical measurement.


Figure 6
View larger version (35K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. {alpha}-TOS-LTVSPWY suppresses breast carcinomas. FVB/N c-neu female mice at 7 to 10 months of age with breast carcinomas, as detected by ultrasound imaging, were treated by i.p injection of the vehicle, 15 or 5 µmol per mouse per dose of {alpha}-TOS, or 5 or 2.5 µmol per mouse per dose of {alpha}-TOS-LTVSPWY ({alpha}-TOS-P). The tumors were visualized and quantified using ultrasound imaging (A) or calipers (B) and are expressed relative to their volume at the onset of the treatment. C, representative images of a tumor from an animal treated with 5 µmol {alpha}-TOS-LTVSPWY at times shown. Points, mean (n = 4–6); bars, SD. *, significant differences between the control animals and animals treated with {alpha}-TOS-peptide at 5 µmol per dose per mouse.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We present here a novel paradigm for cancer therapy based on conjugation of anticancer agents with peptides recognized by cell surface receptors preferentially expressed by malignant cells. The evidence we have provided for this strategy here involves modification of an apoptogenic compound from the group of vitamin E analogues ({alpha}-TOS) with a peptide recognized by the tyrosine receptor kinase erbB2. We show that (a) the conjugate of {alpha}-TOS and the heptapeptide LTVSPWY caused greater levels of apoptosis in erbB2-high than erbB2-low cells; (b) down-regulation of erbB2 partially inhibited {alpha}-TOS-LTVSPWY–induced apoptosis in erbB2-high cells; (c) apoptosis induced by {alpha}-TOS-LTVSPWY in erbB2-high cells was dependent on endocytosis and lysosomal function; and (d) that {alpha}-TOS-LTVSPWY efficiently suppressed the growth of breast carcinomas in c-neu transgenic mice.

Our hypothesis was based on a study by Shadidi and Sioud (23), showing that addition of specific peptides to small molecules rendered them selective for breast cancer cells. Using the phage display approach, they identified a specific heptapeptide (LTVSPWY) whose attachment to the green fluorescent protein caused rapid internalization of the fluorescent protein by breast cancer cells expressing erbB2. We document here that {alpha}-TOS-LTVSPWY was more rapidly endocytosed by erbB2-overexpressing cells than by erbB2-low cells.

Shadidi and Sioud (23) presented data from experiments using the peptide targeting the erbB2 antisense oligodeoxynucleotides, which resulted in specific down-regulation of the receptor. Supported by our results, we present an intriguing paradigm for cancer therapy because this approach can be used for targeting of divergent molecules to cells overexpressing specific surface receptors. Molecules delivered by this approach include antisense oligodeoxynucleotides, ribozymes, and siRNAs, the targeting of which may be used in treatment of different pathologies without adversely affecting healthy tissue (38–42). There are reports that describe targeting of cytotoxic drugs to tumor cells encapsulated in carriers, including peptide-coated liposomes, as documented in both in vitro and in vivo models (43, 44).

Our results are consistent with those of Song et al. (41), who showed that an erbB2 single-chain antibody fused with protamine targeted erbB2 siRNA preferentially to erbB2-overexpressing breast carcinoma cells. We present data showing that erbB2-high breast cancer cells are more prone to killing by {alpha}-TOS-LTVSPWY than their erbB2-low counterparts. Thus, the difference in the levels of the receptor determines, at least to some extent, the susceptibility of the cells to the peptide conjugate of {alpha}-TOS. The erbB2-low MCF-7 underwent apoptosis when challenged with {alpha}-TOS-LTVSPWY, albeit at a lower rate than did erbB2-high cells, most probably reflecting the greatly reduced levels of the receptor in these cells (45), which results in reduced binding and uptake of the peptide conjugate. Hence, the level of expression of erbB2 was shown to provide selective killing by the peptide conjugate, and we can expect that normal cells, with low erbB2 expression, will not be killed by the peptide-vitamin E analogue conjugate.

Our results with cells in which endocytosis was blocked by overexpression of DN dynamin-2 suggest that internalization of {alpha}-TOS-LTVSPWY by breast cancer cells is dependent on endocytosis via clathrin-coated vesicles because dynamin-2 is essential for the formation and release of these vesicles and their conversion into endosomes (29). Therefore, it can be postulated that after binding to the erbB2 receptor, the peptide with the drug is endocytosed. In the mildly acidic late endosome, the receptor is detached from the {alpha}-TOS-peptide conjugate, which travels to the lysosome, where acidic peptidases liberate free {alpha}-TOS from the peptide conjugate. This is an important event in the peptide-{alpha}-TOS processing by the cell because free carboxylate is required for the proapoptotic activity of {alpha}-TOS (36, 37). Free vitamin E succinate then induces apoptosis by targeting mitochondria, leading to ROS generation and dissipation of {Delta}{Psi}m followed by activation of caspases and entry of the cell to the commitment phase of apoptosis (cf. Fig. 2; refs. 26, 35).

Importantly, efficient uptake of the peptide conjugate of {alpha}-TOS results in pronounced suppression of breast carcinomas, as documented by experiments with c-neu transgenic mice expressing high levels of erbB2 in the mammary epithelial cells (32). Data in Fig. 6 reveal that the peptide conjugate was more efficient than when {alpha}-TOS alone was used at thrice higher doses. The superiority of the peptide conjugate may be due to both greater selective uptake by the erbB2-high cancer cells and greater efficacy of the conjugate to deliver free {alpha}-TOS inside the cells, resulting in rapid apoptosis.

In summary, we present in this report an intriguing paradigm for cancer therapy, according to which an inducer of apoptosis, epitomized by {alpha}-TOS, is modified by addition of the peptide LTVSPWY. The conjugate is rapidly endocytosed by erbB2-overexpressing cells and internalized within endosomes. Their maturation into lysosomes results in hydrolysis of {alpha}-TOS-LTVSPWY by the action of acidic peptidases. The liberated {alpha}-TOS, a potent mitocan (46, 47), then compromises the mitochondrial function via generation of ROS and dissipation of {Delta}{Psi}m. This leads to activation of the mitochondria-specific caspase-9 and entry of the cell into the apoptosis commitment phase. This approach, based on targeting of anticancer drugs to malignant cell, may be of clinical relevance.


   Acknowledgments
 
Grant support: Australian Research Council, Queensland Cancer Fund, National Breast Cancer Foundation (J. Neuzil), The Grant Agency of the Ministry of Agriculture of Czech Republic (grant QF 3115/2003) and the Institutional Research Concept for the Institute of Organic Chemistry and Biochemistry grant AZ4 055 0506 (M. Ledvina).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Youle (National Institutes of Health, Bethesda, MD) for providing the Bcl-xL plasmid, Dr. Takei (Juntendo University School of Medicine, Tokyo, Japan) for providing the dynamin-2 plasmids, Dr. Schiff (Baylor College of Medicine, Houston, TX). for providing the MCF-7/HER2-18 cells, Dr. Smith (Otago University, Dunedin, New Zealand). for providing the MitoQ, Dr. Witting (ANZAC Research Institute, Concorde, NSW, Australia) for providing the LDL, and Dr. Sioud (Norwegian Radium Hospital, Oslo, Norway) for critical reading of the article.


   Footnotes
 
Note: L.H. Yuan is a visiting student at the Griffith University, Southport, Queensland, Australia.

This work forms a part of the Ph.D. thesis of X-F. Wang.

Received 7/ 6/06. Revised 1/ 2/07. Accepted 1/30/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Roskoski R. The ErbB/HER receptor protein-tyrosine kinases and cancer. Biochem Biophys Res Commun 2002;319:1–11.[CrossRef]
  2. Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989;244:707–12.[Abstract/Free Full Text]
  3. Zhou BP, Hung MC. Dysregulation of cellular signaling by HER2/neu in breast cancer. Semin Oncol 2003;30:38–48.[Medline]
  4. Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat Rev Cancer 2002;2:489–501.[CrossRef][Medline]
  5. Dudek H, Datta SR, Franke TF, et al. Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 1997;275:661–5.[Abstract/Free Full Text]
  6. Cardone MH, Roy N, Stennicke HR, et al. Regulation of cell death protease caspase-9 by phosphorylation. Science 1998;282:1318–21.[Abstract/Free Full Text]
  7. Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 1997;9:231–41.
  8. Xin M, Deng X. Nicotine inactivation of the proapoptotic function of Bax through phosphorylation. J Biol Chem 2005;280:10781–9.[Abstract/Free Full Text]
  9. Kane LP, Shapiro VS, Stokoe D, Weiss A. Induction of NF-{kappa}B by the Akt/PKB kinase. Curr Biol 1999;9:601–4.[CrossRef][Medline]
  10. LaCasse EC, Baird S, Korneluk RG, MacKenzie AE. The inhibitors of apoptosis (IAPs) and their emerging role in cancer. Oncogene 1998;7:3247–59.
  11. Fukuda S, Foster RG, Porter SB, Pelus LM. The antiapoptosis protein survivin is associated with cell cycle entry of normal cord blood CD34(+) cells and modulates cell cycle and proliferation of mouse hematopoietic progenitor cells. Blood 2002;100:2463–71.[Abstract/Free Full Text]
  12. Mitsiades CS, Mitsiades N, Poulaki V, et al. Activation of NF-{kappa}B and upregulation of intracellular anti-apoptotic proteins via the IGF-1/Akt signaling in human multiple myeloma cells: therapeutic implications. Oncogene 2002;21:5673–83.[CrossRef][Medline]
  13. Neuzil J, Tomasetti M, Mellick AS, et al. Vitamin E analogues: a new class of inducers of apoptosis with selective anti-cancer effect. Curr Cancer Drug Targets 2004;4:267–84.[CrossRef][Medline]
  14. Wang XF, Dong LF, Zhao Y, Tomasetti M, Wu K, Neuzil J. Vitamin E analogues as anti-cancer agents: lessons from studies with {alpha}-tocopheryl succinate. Mol Nutr Food Res 2006;50:675–85.[CrossRef][Medline]
  15. Neuzil J, Weber T, Gellert N, Weber C. Selective cancer cell killing by {alpha}-tocopheryl succinate. Br J Cancer 2001;84:87–9.[CrossRef][Medline]
  16. Malafa MP, Neitzel LT. Vitamin E succinate promotes breast cancer tumor dormancy. J Surg Res 2000;93:163–70.[CrossRef][Medline]
  17. Malafa MP, Fokum FD, Mowlavi A, et al. Vitamin E inhibits melanoma growth in mice. Surgery 2002;131:85–91.[CrossRef][Medline]
  18. Weber T, Lu M, Andera L, et al. Vitamin E succinate is a potent novel anti-neoplastic agent with high tumor selectivity and cooperativity with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2L) in vivo. Clin Cancer Res 2002;8:863–9.[Abstract/Free Full Text]
  19. Tomasetti M, Gellert N, Procopio A, Neuzil J. A vitamin E analogue suppresses malignant mesothelioma in a pre-clinical model: a prototype of a future drug against a fatal neoplastic disease? Int J Cancer 2002;109:641–2.[CrossRef]
  20. Stapelberg M, Gellert N, Swettenham E, et al. {alpha}-Tocopheryl succinate inhibits malignant mesothelioma by disruption of the FGF autocrine signaling loop: mechanism and the role of oxidative stress. J Biol Chem 2005;280:25369–76.[Abstract/Free Full Text]
  21. Wang XF, Witting PK, Salvatore BA, Neuzil J. {alpha}-Tocopheryl succinate induces apoptosis in HER2/erbB2-overexpressing breast cancer cells by signalling via the mitochondrial pathway. Biochem Biophys Res Commun 2005;326:282–9.[CrossRef][Medline]
  22. Neuzil J, Zhao M, Ostermann G, et al. {alpha}-Tocopheryl succinate, an agent with in vivo anti-tumour activity, induces apoptosis by causing lysosomal instability. Biochem J 2002;362:709–15.[CrossRef][Medline]
  23. Shadidi M, Sioud M. Identification of novel carrier peptides for the specific delivery of therapeutics into cancer cells. FASEB J 2003;17:256–8.[Abstract/Free Full Text]
  24. Shou J, Massarsweh S, Osborne CK, Wakelong AE, Ali S, Schiff R. Mechanism of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer. J Natl Cancer Inst 2004;96:926–35.[Abstract/Free Full Text]
  25. Kurokawa H, Lenferink AE, Simpson JF, et al. Inhibition of HER2/neu (erbB-2) and mitogen activated protein kinases enhances tamoxifen action against HER2-overexpressing, tamoxifen-resistant breast cancer cells. Cancer Res 2000;60:5887–94.[Abstract/Free Full Text]
  26. Weber T, Dalen H, Andera L, et al. Mitochondria play a central role in apoptosis induced by {alpha}-tocopheryl succinate, an agent with anticancer activity. Comparison with receptor-mediated pro-apoptotic signaling. Biochemistry 2003;42:4277–91.[CrossRef][Medline]
  27. Swettenham E, Witting PK, Salvatore BA, Neuzil J. {alpha}-Tocopheryl succinate selectively induces apoptosis in neuroblastoma cells: potential therapy of malignancies of the nervous system? J Neurochem 2005;94:1448–56.[CrossRef][Medline]
  28. Kelso GF, Porteous CM, Coulter CV, et al. Selective targeting of a redox-active ubiquinone to mitochondria within cells: antioxidant and antiapoptotic properties. J Biol Chem 2001;276:4588–96.[Abstract/Free Full Text]
  29. Wolter KG, Hsu YT, Smith CL, Nechushtan A, Xi XG, Youle RJ. Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol 1997;139:1281–92.[Abstract/Free Full Text]
  30. Kashiwakura Y, Watanabe M, Kusumi N, et al. Dynamin-2 regulates oxidized low-density lipoprotein-induced apoptosis of vascular smooth muscle cell. Circulation 2004;110:3329–34.[Abstract/Free Full Text]
  31. Jostarndt K, Rubic T, Kühn H, et al. Enzymatically modified LDL upregulates CD36 expression in non-differentiated monocytic cells in a PPAR-{gamma}-dependent mode. Biochem Pharmacol 2004;67:841–54.[CrossRef][Medline]
  32. Guy CT, Webster MA, Schaller M, Parsons TJ, Cardiff RD, Muller WJ. Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease. Proc Natl Acad Sci U S A 1992;89:10578–82.[Abstract/Free Full Text]
  33. Fariss MW, Merson MH, O'Hara TM. {alpha}-Tocopheryl succinate protects hepatocytes from chemical-induced toxicity under physiological calcium conditions. Toxicol Lett 1989;47:61–75.[CrossRef][Medline]
  34. Neuzil J, Weber T, Schröder A, et al. Induction of apoptosis in cancer cells by {alpha}-tocopheryl succinate: molecular pathways and structural requirements. FASEB J 2001;15:403–15.[Abstract/Free Full Text]
  35. Yu W, Sanders BG, Kline K. RRR-{alpha}-Tocopheryl succinate-induced apoptosis of human breast cancer cells involves Bax translocation to mitochondria. Cancer Res 2003;63:2483–91.[Abstract/Free Full Text]
  36. Birringer M, EyTina JH, Salvatore BA, Neuzil J. Vitamin E analogues as inducers of apoptosis: structure-function relationship. Br J Cancer 2003;88:1948–55.[CrossRef][Medline]
  37. Tomic-Vatic A, EyTina JH, Chapmann JM, Mahdavian E, Neuzil J, Salvatore BA. Vitamin E amides, a new class of vitamin E analogues with enhanced pro-apoptotic activity. Int J Cancer 2005;117:188–93.[CrossRef][Medline]
  38. Gewirtz AM, Sokol DL, Ratajczak MZ. Nucleic acid therapeutics: state of the art and future perspectives. Blood 1998;92:712–36.[Free Full Text]
  39. Shadidi M, Sioud M. Selective targeting of cancer cells using synthetic peptides. Drug Resist Updat 2003;6:363–71.[CrossRef][Medline]
  40. Stefanidakis M, Koivunen E. Peptide-mediated delivery of therapeutic and imaging agents into mammalian cells. Curr Pharm Des 2004;10:3033–44.[CrossRef][Medline]
  41. Song E, Zhu P, Lee S, et al. Antibody mediated in vivo delivery of small interfering RNA via cell-surface receptors. Nat Biotechnol 2005;23:709–17.[CrossRef][Medline]
  42. Sauer I, Dunay IR, Weisgraber K, Bienert M, Dathe M. An apolipoprotein E-derived peptide mediates uptake of sterically stabilized liposomes into brain capillary endothelial cells. Biochemistry 2005;44:2021–9.[CrossRef][Medline]
  43. Dharap SS, Wang Y, Chandna P, et al. Tumor-specific targeting of anticancer drug delivery system by LHRH peptide. Proc Natl Acad Sci U S A 2005;102:12962–7.[Abstract/Free Full Text]
  44. Arap W, Pasqualini R, Ruoslahti E. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 1998;279:377–80.[Abstract/Free Full Text]
  45. Karunagaran D, Tzahar E, Beerli RR, et al. ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer. EMBO J 1996;15:254–264.[Medline]
  46. Ralph SJ, Dong LF, Low P, Lawen A, Neuzil J. Mitocans: mitochondria-targeted anti-cancer drugs as improved therapies and related patents. Recent Patent Reviews Anti-Cancer Drug Discovery 2006;1:327–46.
  47. Neuzil J, Wang XF, Dong LF, Low P, Ralph SJ. Molecular mechanism of ‘mitocan’-induced apoptosis in cancer cells epitomizes the multiple roles of reactive oxygen species and Bcl-2 family proteins. FEBS Lett 2006;580:5125–9.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 CarcinogenesisHome page
P.-H. Huang, D. Wang, H.-C. Chuang, S. Wei, S. K. Kulp, and C.-S. Chen
{alpha}-Tocopheryl succinate and derivatives mediate the transcriptional repression of androgen receptor in prostate cancer cells by targeting the PP2A-JNK-Sp1-signaling axis
Carcinogenesis, July 1, 2009; 30(7): 1125 - 1131.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
L.-F. Dong, R. Freeman, J. Liu, R. Zobalova, A. Marin-Hernandez, M. Stantic, J. Rohlena, K. Valis, S. Rodriguez-Enriquez, B. Butcher, et al.
Suppression of Tumor Growth In vivo by the Mitocan {alpha}-tocopheryl Succinate Requires Respiratory Complex II
Clin. Cancer Res., March 1, 2009; 15(5): 1593 - 1600.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
X. Gu, X. Song, Y. Dong, H. Cai, E. Walters, R. Zhang, X. Pang, T. Xie, Y. Guo, R. Sridhar, et al.
Vitamin E Succinate Induces Ceramide-Mediated Apoptosis in Head and Neck Squamous Cell Carcinoma In vitro and In vivo
Clin. Cancer Res., March 15, 2008; 14(6): 1840 - 1848.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
L.-F. Dong, E. Swettenham, J. Eliasson, X.-F. Wang, M. Gold, Y. Medunic, M. Stantic, P. Low, L. Prochazka, P. K. Witting, et al.
Vitamin E Analogues Inhibit Angiogenesis by Selective Induction of Apoptosis in Proliferating Endothelial Cells: The Role of Oxidative Stress
Cancer Res., December 15, 2007; 67(24): 11906 - 11913.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, X.-F.
Right arrow Articles by Neuzil, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, X.-F.
Right arrow Articles by Neuzil, J.

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