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Keywords:

  • parathyroid hormone-related protein 38–94;
  • invasion index;
  • cell viability;
  • nude mice

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Parathyroid hormone-related protein (PTHrP) is critical for normal mammary development and is overexpressed by breast cancers. PTHrP is a peptide hormone that undergoes extensive post-translational processing, and PTHrP(38–94)-amide is one of the mature secretory forms of the peptide. In this study, we explored the effect of PTHrP(38–94)-amide in a panel of six breast cancer cell lines “in vitro” and in MDA-MB231 cells “in vivo” specifically examining cell viability, proliferation, invasiveness, and growth in nude mice. PTHrP(38–94)-amide markedly inhibited proliferation and also caused striking toxicity and accelerated cell death in breast cancer cells. In addition, direct injection of PTHrP(38–94)-amide into MDA-MB231 breast cancer cells passaged in immunodeficient mice produced a marked reduction in tumor growth. These studies (i) indicate breast cancer cells are one of the few tissues in which specific effects of midregion PTHrP have been established to date, (ii) support a role for midregion secretory forms of PTHrP in modulating not only normal but also pathological mammary growth and differentiation, (iii) add further evidence for the existence of a specific midregion PTHrP receptor, and (iv) provide a novel molecule for modeling of small molecule analogues that may have anti-breast cancer effects.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

PARATHYROID HORMONE-RELATED protein (PTHrP) was first described in 1987 as the factor responsible for the large majority of instances of humoral hypercalcemia of malignancy. More recently, a great deal of experimental evidence has established that in addition to its pathophysiological role in cancer, PTHrP is produced normally in most normal tissues throughout the body.(1) One of the tissues that produces PTHrP is breast epithelium, and PTHrP concentrations are markedly elevated in normal human and rodent milk.(2, 3) Although the precise functions of PTHrP within the lactating breast remain incompletely defined, it is now very clear that PTHrP is a critical factor in normal mammary development.(4, 5) For example, disruption of the PTHrP gene in mice leads to the complete failure of mammary development in both male and female fetuses.(6) Interestingly, humans with inactivating mutations of the PTH/PTHrP receptor also display complete failure of mammary gland or nipple development.(7) Conversely, targeted overexpression of PTHrP in the mammary epithelium in transgenic mice leads to the development of a hypoplastic mammary ductular system, with inadequate fat pad penetration, incomplete branching morphogenesis, failure of lactation, and resultant death of offspring.(8) Thus, in normal mammary development, both timing (embryonal vs. neonatal vs. adolescent vs. lactational expression) and location (nipple rudiment vs. ductular epithelium) of PTHrP production are critical to mammary development and function both in rodents and in humans.

One of the original tumors from which PTHrP was purified and sequenced, because of the elevated concentration of its circulating form, was breast cancer,(9) and it is now well-known that PTHrP is involved centrally in the cancer-associated hypercalcemic syndrome resulting from systemic overproduction of PTHrP(10) and its local production within skeletal metastases with the following activation of bone resorption.(11) Interestingly, several authors have indicated that expression of PTHrP is higher and more frequent in breast cancer skeletal metastases than in the primary tumor from which these were derived.12-14) Indeed, PTHrP has been implicated both in the osteotropism of breast cancer as well as in the breast cancer-mediated bone resorption.(11) Finally, specific patterns of PTHrP promoter usage and alternative splicing events in breast cancer, as compared with other tissues and tumors, has been suggested from both “in vitro” and “in vivo” model systems.15-17) Thus, the ties between breast cancer and PTHrP are extensive.

PTHrP is a prohormone, the post-translational processing of which generates a family of mature secretory forms of the peptide.(18) One of these is PTHrP(1-36), which contains homology with PTH and is able to activate the PTH/PTHrP receptor. A C-terminal form of PTHrP, termed “osteostatin” and surmised to be comprised of PTHrP(107-139), also has been identified, although its precise natural structure or sequence have not been defined.(19) In addition, a midregion form, that is, PTHrP(38-94), also has been identified and structurally defined; the latter does not act through the PTH/PTHrP receptor but is able to activate intracellular Ca2+ pathways in concentrations as low as 1 pM.(20) In addition, it has been shown to be critical for driving transplacental Ca2+ transport from the maternal to the fetal circulation. However, in contrast to the extensive list of tissues in which a physiological role for PTHrP(1-36) has been characterized, PTHrP(38-94) has been shown to exert an effect in only a few tissues (including placenta and kidney) and cell lines.

Studies examining the effects of PTHrP on the growth and invasiveness of breast cancer cell lines generally have been limited to either N-terminal species or full-length unprocessed PTHrP introduced by gene transfer methods.(21, 22) The one exception to this is a previous report from our groups, before the characterization and synthesis of PTHrP(38-94),(20) which indicated that a synthetic midregion PTHrP fragment, that is, (67-86), and a C-terminal synthetic PTHrP form, that is, (107-139), inhibited proliferation and enhanced invasion in vitro of a single breast cancer cell line.(23)

In this study, our goal was to examine in a comprehensive fashion the effects of the midregion PTHrP secretory form on multiple breast tumor cell lines with respect to growth and invasiveness in vitro, and also tumor growth in vivo. To our surprise, the results indicate that this PTHrP domain is a potent inhibitor of breast cancer growth and invasive tendency both in vitro and in vivo.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Cell cultures and PTHrP fragment

8701-BC,(24) Hs578T, and MDA-MB231 cells were from laboratory stocks, and T47D cells were purchased from ICLC (Genova, Italy). Two clonal lines isolated from parental 8701-BC cells (BC-61 and BC-3A) were also tested as prototypes of more and less tumorigenic cell subpopulations, respectively, in light of their different doubling time in vitro,(25) frequency of tumor development as well as average volume reached in nude mouse.(26) 8701-BC, MDA-MB231, BC-3A, and BC-61 cells were grown in 10% fetal calf serum (FCS) containing RPMI 1640 medium (Gibco, Paisley, UK) plus 100 U of penicillin and 100 μg of streptomycin/ml. T47D cells were grown in the same medium supplemented with 10−7 M of bovine insulin and 2 mM of L-glutamine. Hs578T cells were grown in 10% FCS containing high glucose Dulbecco's modified Eagle's medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with antibiotics, 10−7 M of bovine insulin, and 2 mM of L-glutamine.

PTHrP(38-94)-amide was synthesized using solid phase methods and subjected to amino acid composition, mass spectroscopy, and analytical reversed-phase high-performance liquid chromatography (HPLC) analyses for confirmation of the structure, purity, and peptide content of the preparation.(20)

In vitro proliferation assay

Cell proliferative behavior in response to PTHrP(38-94)-amide was studied as reported previously.(23, 27) Cells were plated at 2.5 × 104 cells/cm2 in the appropriate 10% FCS-containing medium. After overnight incubation to allow adhesion, cells were cultured in serum-free RPMI medium for an additional 24 h; subsequently, serum-free RPMI medium supplemented with PTHrP(38-94)-amide at various concentrations was added to the cultures. After 24 h, fresh medium with the same peptide supplement was added and the cells were incubated for a further 24 h, trypsinized, and counted in a Bürker chamber.

Control assays were performed in the absence of PTHrP(38-94)-amide or in the presence of PTHrP(37-64), which had been proven to be inactive in our model system.(23)

Neutral red uptake assay

MDA-MB231 cells plated in 4-well dishes at 5-10 × 104 cells/well were grown in serum-free medium as described and treated for 3 h with neutral red-containing medium, plain or supplemented with either 100 pM or 1 nM of PTHrP(38-94)-amide. At the end of the incubation, the cells were fixed with 4% formaldehyde plus 1% CaCl2 for 1 minute after which 0.2 ml of solubilization fluid (1% glacial acetic acid in 50% ethanol) was added to each well for 15 minutes under gentle agitation. The absorbance of the solubilized neutral red was read at 540 nm.(28)

[3H]thymidine uptake assay

MDA-MB231 cells, plated in 4-well dishes at 2 × 105 cells/well, were grown in serum-free medium as described and treated with either 100 pM or 1 nM of PTHrP(38-94)-amide for 5, 20, or 40 minutes or 1, 3, or 6 h. Control cells received only RPMI 1640 medium. At the end of every incubation, the medium was removed and substituted with fresh serum-free medium containing 1 μCi/ml of [3H]thymidine (ICN, Thame, UK) and cells were incubated for additional 24 h. Subsequently, they were washed thoroughly with phosphate-buffered saline (PBS) and lysed for 3 minutes with NCS tissue solubilizer (ICN); the radioactivity of the solution was counted with BetaMax scintillation mixture (ICN).

Fluorescence microscopy

Acridine orange and ethidium bromide were dissolved in PBS and added to the culture medium of control and 1 nM of PTHrP(38-94)-amide-treated MDA-MB231 cells at 2 μg/ml of the final concentration. The cells were viewed immediately under either fluorescein isothiocyanate (FITC; for acridine orange) or tetramethylrhodamine isothiocyanate (TRITC; for ethidium bromide) fluorescence and photographed.(29)

In vitro chemotaxis and chemoinvasion assays

Cell chemotactic and chemoinvasive behaviors were evaluated by a modified Boyden chamber test(30) using polyvinylpyrrolidone-free polycarbonate filters with an 8-μm pore diameter (Nucleopore, Pleasanton, CA, USA), and “blind well” chambers with an upper compartment of 800 μl and a lower compartment of 200 μl (Neuro-Probe, Cabin John, MD, USA), as already reported.(23, 27) For chemotaxis assay, trypsinized cells were washed first with 10% FCS-RPMI for enzyme inactivation and then twice with 0.1% bovine serum albumin (BSA)-RPMI medium, and 3 × 105 cells were seeded in the upper compartment of each chamber. PTHrP(38-94)-amide was dissolved at various concentrations in 0.1% BSA-containing medium and placed in the lower compartment of the chamber. The assay was carried out for 6 h; the cells attached to the upper surface of the filter were removed mechanically, whereas those migrated to the lower surface of the filter were fixed with ethanol, stained with toluidine blue, and quantitated by counting the number of cells present in 10-15 random fields of the filter at a 200-fold magnification.

The chemoinvasion assay followed the procedures used in the chemotaxis assay, with the exception that filters were coated with 25 μg of Matrigel, a reconstituted basement membrane matrix from Engelbreth Holm-Swarm (EHS) sarcoma (Collaborative Research, Bedford, MA, USA). Control assays were performed as for the proliferation test.

Invasion parameters were calculated by the following formulas: equation image where I.I. was the invasion index and NCI and NCT were the number of cells crossing the filters in response to the same chemoattractant in parallel chemoinvasion and chemotaxis assays, respectively.

equation image where I.I.t and I.I.c were the I.I. of treated and control cells, being relative I.I. (R.I.I.) values of 1, <1, and >1, respectively, indicative of an invasion-ineffective, -restraining, and -promoting effect exerted by the tested molecule.

In vivo studies

MDA-MB231 cells were cultured under standard conditions and after mild trypsinization and washing, they were resuspended at 2 × 107 cells/ml of serum-free medium and incubated at 37°C for 20 minutes to allow cell recovery. PTHrP(38-94)-amide was dissolved at 1 mg/ml in 10 mM of acetic acid and aliquots were maintained at −20°C. For in vivo studies, the peptide was diluted in sterile PBS at 800 ng/ml (126 nM). The biological activity was assessed by injecting a 40-ng dose daily.(20)

Female athymic BALB/c nude mice (8 weeks old) were obtained from Harlan Ibérica (Barcelona, Spain) and housed in a laminar-flow cabinet under specific pathogen-free conditions and under protocol of the Laboratory Animal Care of the Complutense University of Madrid/E. Nude mice received water and food ad libitum and were kept with a daily photoperiod of 12 h light and 12 h dark. The in vivo effect of the PTHrP(38-94)-amide on the tumorigenic capacity of MDA-MB231 cells was tested by orthotopic injection of the cells into the mammary fat pad (m.f.p.) of nude mice, as previously described.(31) Tumor growth was initiated by injection of 50 μl of cell suspension (106 cells/site) into the m.f.p. The mice were surveyed daily to determine when tumors first became apparent on visual inspection and then tumor sizes were measured weekly. Tumor size was calculated by measuring the dimensions of the tumor mass (average of two right-angle diameters, the largest and its perpendicular) with a vernier caliper.(32) The percentage of tumor size variation (Δ%) was calculated using the following formula: equation image where St was the tumor size at each time point and S0 was the tumor size at time 0.

Three weeks after inoculation of the cells, two groups of 6 mice each were given a daily intratumor injection of 50 μl of PBS for the control group and 50 μl of PTHrP(38-94)-amide (40 ng) for the treated group and tumor growth was monitored every day. In a second set of experiments (6 mice), MDA-MB231 tumors were induced in both flanks of each mouse and then the m.f.p. of one flank of the animal was injected with PBS and the other flank was injected with the peptide. The treatment and tumor monitoring were performed as described previously. Tumor-bearing mice were killed on day 14 after the beginning of the peptide/vehicle treatment and tumor specimens were removed and weighed.

Statistics

The results from in vitro studies are presented as mean ± SEM of three different experiments, each performed in quadruplicate; the SEM is indicated as vertical bars in the figures. Data were analyzed using software-assisted analysis of variance (ANOVA) (SigmaStat v.2; SPSS, Chicago, IL, USA) and p < 0.05 was taken as the minimal level of statistical significance.

In the in vivo experiments, the normal distribution of the size among the different tumors belonging to the same group at different time points was determined by the Kolmogorov-Smirnov test. Complex ANOVA analysis with repeated measurements (SPSS 10.0.7; SPSS) was performed to determine if there were significant differences between the tumor size evolution among the two experimental groups and to establish which time point on the data were significantly different.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Selection and characterization of the breast cancer cell lines for the in vitro studies

Six breast cancer cell lines (T47-D, MDA-MB231, Hs578T, 8701-BC, and the two derived clones BC 61 and BC-3A) were used; their selection was based on their representing the spectrum of breast cancers encountered in medical practice. They span the range of histochemical subtypes of breast cancer, estrogen receptor status, vimentin expression, activity in chemotaxis and chemoinvasion assays, and invasive behavior in nude mice.(33, 34) Table 1 provides a summary of the biological characteristics of the breast cancer cell lines. By the criteria in the table, the parental cell lines can be subdivided into three groups, displaying high (Hs578T and MDA-MB231), intermediate (8701-BC), and low neoplastic “aggressiveness” (T47D).

Table Table 1.. Biological Characteristics of Human Breast Cancer Cell Lines
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PTHrP(38-94)-amide restrains breast cancer cell number in vitro

Other N-terminal and full-length PTHrP species have been reported previously to influence the proliferation of individual breast cancer cell lines.(21, 22) Because our preliminary findings indicated that PTHrP(67-86) could inhibit the proliferation of 8701-BC cells,(23) we first wanted to examine comprehensively the effects of midregion PTHrP(38-94)-amide on a broad panel of representative breast cancer cell lines. The results are shown in Fig. 1.

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Figure FIG. 1. Growth response of breast cancer cell lines to PTHrP(38-94)-amide (•) and PTHrP(37-64) (○) at different concentrations. *p < 0.05; **p < 0.001.

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Three observations emerge from this figure. First, PTHrP(38-94)-amide inhibits cell growth in each of the cell lines, with a maximum effect at concentrations of approximately 1 nM and with initial effects at doses as low as 1-10 pM. Second, the effects of midregion PTHrP on Hs578T and T47-D cells are biphasic, with a loss or reversal of effect as doses rise into the 10-nM range. Third, the two subclones of 8701-BC, that is, BC-61 and BC-3A, displayed different responses than those observed in the parental 8701-BC cells, with the antiproliferative effect of PTHrP being quantitatively weaker and the dose-response curve displaying a different shape, achieving responses at a lower concentrations than that required by the parental line. This last result provides additional evidence for the existence of intratumoral heterogeneity for response to PTHrP within the composite 8701-BC cell line.(24, 27) Finally, in Hs578T and T47-D cells PTHrP(38-94)-amide appeared to increase cell number at very low doses (1 pM and 10 pM).

PTHrP(38-94)-amide reduces breast cancer cell invasion in vitro

Next, we examined the effect of midregion PTHrP on invasiveness of all six cell lines using standard chemotaxis and chemoinvasion assays in modified Boyden chambers, thereby examining the R.I.I. of the six cell lines at different PTHrP concentrations. As shown in Fig. 2, PTHrP(38-94)-amide inhibited the invasiveness of five of the six cell lines. As observed in the growth curves in Fig. 2, the anti-invasiveness maximal dose appeared to be approximately 1 nM, and the anti-invasive effect on Hs578T, MDA-MB231, BC-61, and T47-D cells appeared biphasic, being reversed by higher doses of the peptide. In BC-3A, T47-D, and BC-61 cells, low doses appeared to stimulate invasiveness.

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Figure FIG. 2. R.I.I. of human breast cancer cells in response to PTHrP(38-94)-amide. The SEM of BC-3A cells at 10 nM PTHrP(38-94)-amide is ±1.40. *p < 0.05; **p < 0.001.

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PTHrP(38-94)-amide affects the viability of MDA-MB231 cells

The decline in cell number produced by midregion PTHrP could result from a reduction in the rate of cell proliferation, from acceleration of cell death, or a combination of both of these effects. To obtain additional information, we submitted MDA-MB231 cells, which were the most responsive to PTHrP inhibitory influence, to a series of cell viability tests with different endpoints.

First, we examined the rate of [H3]thymidine incorporation by cells after different times of incubation with PTHrP(38-94)-amide. Figure 3 shows that addition of 100 pM or 1 nM of PTHrP(38-94)-amide to MDA-MB231 cells reduced subsequent [H3]thymidine uptake by 30-50% in an exposure time-dependent manner within 40-60 minutes. Longer periods of treatment up to 6 h yielded no additional inhibitory effect.

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Figure FIG. 3. Percent of [H3]thymidine uptake by MDA-MB231 cells after different times of 100 pM (•) and 1 nM (○) PTHrP(38-94)-amide treatments versus control. *p < 0.05; **p < 0.001

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Then, we chose to examine the uptake of neutral red, a vital dye that accumulates in the lysosomes of live cells only, by MDA-MB231 cells after the addition of increasing doses of PTHrP(38-94)-amide. Figure 4 shows that midregion PTHrP is indeed cytotoxic for MDA-MB231 cells, reducing viability by approximately 40% under the experimental conditions used. As is clear from the study, also, this effect is biphasic and closely parallels in dose the cell number effect observed in Fig. 1.

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Figure FIG. 4. Percent of neutral red uptake by MDA-MB231 cells in the presence of various concentrations of PTHrP(38-94)-amide versus control. *p < 0.05; **p < 0.001

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In a third set of experiments, we examined simultaneous ethidium bromide and acridine orange staining in live MDA-MB231 cells. Ethidium bromide is unable to penetrate the intact membrane of live cells thereby representing a morphological marker for detection of cell death, whereas acridine orange, which permeates cells freely, serves as a counterstain for visualization of total cells in the microscopic field. Figure 5 shows that 1 nM of PTHrP(38-94)-amide induces a marked increase in the number of ethidium bromide-positive MDA-MB231 cells versus control.

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Figure FIG. 5. Fluorescence micrographs of control and PTHrP(38-94)-treated MDA-MB231 cells stained with acridine orange (AO) and ethidium bromide (EB; magnification ×40).

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These findings collectively indicate that in addition to its antiproliferative effect, PTHrP(38-94)-amide is cytotoxic for MDA-MB231 cells and induces cell death via a nonapoptotic pathway.

PTHrP(38-94)-amide reduces the tumorigenesis of MDA-MB231 cells in vivo

Next, we examined the effects of midregion PTHrP on the growth of MDA-MB231 tumors in vivo. For these studies, we injected 106 MDA-MB231 cells into the m.f.p. of 12 nude mice. Three weeks after tumor injection, the average diameter of the tumors was 0.5-0.7 cm. Beginning at this time point (time 0 in Fig. 6), 6 mice received a daily intratumor injection of 40 ng of midregion PTHrP in PBS, and the other 6 mice received PBS alone. Figure 6A shows the effect of daily injections of PBS or PTHrP on tumor size in the most responsive mouse to peptide injection. As can be seen in the figure, midregion PTHrP treatment decreased the tumor growth rate from 0.37 to 0.08 mm/day (mean reduction from 0.38 ± 0.06 mm/day to 0.26 ± 0.11 mm/day; p = 0.032).

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Figure FIG. 6. Growth of control (•) and PTHrP(38-94)-amide-treated (○) MDA-MB231 tumors in nude mice. (A) Data from two mice, one from the control group and the other from the treated group. (B) Data from a mouse bearing tumors in each flank, one receiving PBS and the other receiving PTHrP. Tumor size is plotted versus the time after the beginning of daily PTHrP treatment. (C) Calculation of tumor size variation at each time point as the percentage of size change (Δ%/day), referred to the size of the tumor at the beginning of the treatment (day 0). Data are presented as mean ± SD. ANOVA analysis reveals significant differences (*p < 0.05) between the experimental groups at day 5 and day 6 and highly significant ones (**p < 0.001) from then on.

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We also have studied a second group of 6 animals in which both flanks were injected with 106 MDA-MB231 cells. Mice bearing tumors in both m.f.p.'s were then injected with PBS in one m.f.p. and PTHrP(38-94)-amide in the other, as described previously. Figure 6B shows the results obtained with the most responsive mouse of the tested group in which the tumor growth rate is shown to decrease from 0.56 to 0.19 mm/day in the presence of PTHrP (mean reduction from 0.35 ± 0.11 to 0.22 ± 0.02; p = 0.043). Figure 6C summarizes all of the data derived from PBS (n = 12) as compared with midregion PTHrP treatment (n = 12) in all the tumors from the animals studied. The growth reduction of PTHrP-treated tumors appears to be highly significant, with an overall reduction at the end of treatment to 36 ± 22% as compared with 79 ± 21% of control tumors (p = 0.017). ANOVA analysis of the time-dependent variation in tumor size from the two groups and of the data of percent change from time 0 reveals that a significant difference (p < 0.05) in the evolution of tumor size is detectable from the fifth day and becomes highly significant (p < 0.001) from the seventh day on after the injection. The different growth rate was not caused by variations in the initial tumor size because there were no differences between the treated and the control groups (6.01 ± 0.92 mm and 6.25 ± 1.22 mm, respectively; p = 0.60).

These results were confirmed through direct weight measures of excised tumors. As shown visually in Fig. 7, the reductions in tumor size were dramatic. The weights of the tumors surgically excised from mice killed on day 14 after the beginning of the treatment were decreased by 34 ± 25% from 1 ± 0.42 g in the control group (n = 9) to 0.6 ± 0.17 g in the presence of PTHrP(38-94)-amide (n = 9; p = 0.026).

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Figure FIG. 7. Morphological appearance, size and weights of tumors. (A) Tumors from two mice, one from the control group and the other from the PTHrP-treated group. (B) Tumors from a mouse bearing tumors in each flank, one receiving PBS and the other the midregion PTHrP.

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These studies indicate that direct intratumor injection of midregion PTHrP markedly decreases tumor mass in vivo, and that this is not the result of systemic absorption and delivery of the peptide, but instead is a direct intratumor effect.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The biologies of PTHrP, mammary development and physiology, and breast cancer are highly intertwined and complex as summarized in the Introduction. Here, we show that a previously little-studied form of PTHrP, midregion PTHrP(38-94)-amide, has dramatic effects on a broad, representative panel of breast cancer cell lines, inhibiting growth and invasion of these lines both in vitro and in one studied line in vivo. Midregion PTHrP was shown to accomplish this reduction in cell number through a combination of a slowing of proliferation as well as an acceleration of cell death. Importantly, the antitumor effects of PTHrP(38-94)-amide were observed not only in vitro but also in vivo after inoculation of MDA-MB231 cells into immunocompromised mice. Further studies will be required to determine the precise cellular pathway through which PTHrP(38-94)-amide accomplishes its mammary carcinoma cell death and antiproliferative effects. However, these results are important for several reasons.

First, the observation that midregion PTHrP markedly reduced breast cancer growth and invasion in vitro and in vivo has potentially important therapeutic implications. Although there seems little reason to be enthusiastic regarding the potential for repeated daily injections of antitumor compounds in humans, the potential that midregion PTHrP might serve as a molecular model for smaller, orally active analogues merits consideration; this molecule represents a unique and novel target for molecular modeling of antineoplastic compounds.

Second, although is it clear that PTHrP is a prohormone and that multiple secretory forms of the peptide exist,(1, 18) the current understanding of the physiology and biology of the non-N-terminal forms of the peptide is very limited. To date, midregion PTHrP has been shown to exert effects on a limited number of cell lines and organs, including the placenta, the kidney, vascular smooth muscle cells, and cultured pancreatic islet cells.(20, 35) The demonstration of biological effects on breast cancer cell lines adds a new tissue to the growing group of midregion PTHrP-responsive tissues.

Third, these studies suggest that the physiology of PTHrP in breast development and function may include forms of PTHrP beyond those that have been studied most extensively to date: PTHrP(1-36) and full-length PTHrP.(21, 22) Based on the current observations, midregion PTHrP may seemingly play physiological roles in mammary ductular development, fat pad invasion, branching morphogenesis, and/or lactation. This area needs to be studied further.

Fourth, these studies provide additional evidence for the existence of a midregion PTHrP receptor. To date, several receptors that recognize aminoterminal PTH and PTHrP have been identified, including the mammalian PTH/PTHrP receptor (also termed the PTH-1 receptor) and the PTH-2 receptor. Evidence exists for a third aminoterminal receptor in teleosts, the PTH-3 receptor, as well.(36, 37) All of these three receptors recognize N-terminal forms of PTH and/or PTHrP. In contrast, although there is clear pharmacologic evidence for the existence of a receptor for midregion PTHrP,(38) such a receptor has not been molecularly cloned or purified yet, despite aggressive attempts by several laboratories.(39) Thus, these studies provide additional impetus to identify the midregion PTHrP receptor, particularly in view of its possible role in regulating the growth and invasiveness of mammary carcinoma.

Fifth, these findings suggest that varying levels of expression or production of either midregion PTHrP or the midregion PTHrP receptor could conceivably account, at least to some extent, for the varying degrees of aggressiveness among human breast cancers. The antiproliferative and anti-invasive effects of midregion PTHrP observed in this study may appear to conflict with the previously described osteotropic and bone-resorbing effects of PTHrP-expressing breast carcinoma cells. It is important to bear in mind here that PTHrP expression and function has multiple layers of complexity, with multiple secretory forms, multiple receptors all of which may be present in secreting and responding cells in varying quantities. Moreover, the effects of PTHrP may be paracrine, autocrine, or intracrine. Thus, breast cancer-PTHrP-skeletal interactions are complex, and a complete understanding will require additional investigation.

This study has several limitations. First, the biphasic nature of the responses shown in Figs. 1, 2, and 4 is curious. Is this a reflection of artifactual precipitation or interaction of midregion PTHrP at higher doses? Is this a result of higher doses of PTHrP “cross-talking” with additional receptors coupled to cellular proliferation or antiapoptotic pathways? Is this a reflection of activation of different signaling pathways by different doses of midregion PTHrP? Moreover, most importantly, perhaps, will higher doses of midregion PTHrP and its derivatives actually stimulate breast cancer proliferation? This worrisome possibility is suggested by the apparent stimulation of proliferation observed with 1-10 pM of peptide in Hs578T and T47D cells (Fig. 1). Second, will the effects observed in the six breast carcinoma lines observed here apply to a more complete range of human breast cancer cell lines? The answers to these questions will require further study.

In summary, we have shown that PTHrP(38-94) is a potent inhibitor of breast carcinoma growth in vitro and in vivo. Midregion PTHrP may be an attractive target for molecular modeling for antineoplastic agents in breast cancer, and it merits further study as a regulatory molecule in normal mammary biology and function.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This work was supported by grants from Italian Ministero dell Universita è della Ricerca Scientifica e Tecnologica (MURST) (ex 60% and Cofin) and the University of Palermo (Contributo per Collaborazioni Scientifica Inter-Universitarie) to C.L., by grant PB98-0083 from the Dirección General de Enseñanza Superior (DGES) (Spain) to M.A.L., and by National Institutes of Health (NIH) grants DK 47168, DK 51081, and DK 54308 to A.F.S.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  • 1
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