Research ArticleExperimental Studies

High-fat Diet Enhances and Plasminogen Activator Inhibitor-1 Deficiency Attenuates Bone Loss in Mice with Lewis Lung Carcinoma

LIN YAN, FORREST H. NIELSEN, SNEHA SUNDARAM and JAY CAO
Anticancer Research July 2015, 35 (7) 3839-3847;

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Abstract

This study determined the effects of a high-fat diet and plasminogen activator inhibitor-1 deficiency (Pai1−/−) on the bone structure in male C57BL/6 mice bearing Lewis lung carcinoma (LLC) in lungs. Significant reduction in bone volume fraction (BV/TV), trabecular number (Tb.N) and bone mineral density (BMD) in femurs and vertebrae were found in LLC-bearing mice compared to non-tumor-bearing mice. In LLC-bearing mice, the high-fat diet compared to the AIN93G control diet significantly reduced BV/TV, Tb.N and BMD in femurs and BV/TV in vertebrae. The high-fat diet significantly reduced BMD in vertebrae in wild-type mice but not in Pai1−/− mice. Compared to wild-type mice, PAI1 deficiency significantly increased BV/TV and Tb.N in femurs. The plasma concentration of osteocalcin was significantly lower and that of tartrate-resistant acid phosphatase 5b (TRAP5b) was significantly higher in LLC-bearing mice. The high-fat diet significantly reduced plasma osteocalcin and increased TRAP5b. Deficiency in PAI1 prevented the high-fat diet-induced increases in plasma TRAP5b. These findings demonstrate that a high-fat diet enhances, whereas PAI1 deficiency, attenuates metastasis-associated bone loss, indicating that a high-fat diet and PAI1 contribute to metastasis-associated bone deterioration.

Metastasis, the spread of malignant cells from a primary tumor to different sites of the same organ or to distant organs, is the most devastating aspect of cancer. Its occurrence signifies systemic, progressive and eventually incurable malignancy if it remains untreated. Because of its aggressiveness, metastasis is accompanied by wasting throughout the course of malignant progression, which eventually leads to cachexia characterized by a significant reduction in body weight and multi-organ functional failure that is responsible for the majority of cancer-related deaths. Clearly, the prevention and treatment of metastasis and its associated wasting remain great challenges to cancer research and clinical practice.

Bone, a rigid organ that supports and protects various organs of the body, is adversely affected by cancer-associated wasting. Patients with lung cancer with a 30% loss of body mass exhibit significantly lower total body mineral content than healthy-matched controls (1). Concurrent muscular and bone deterioration has been reported in a murine model of cancer cachexia (2). It has been proposed that muscle and bone mass may be regulated in tandem during cachexia because many signaling pathways, that induce muscular wasting, also promote bone loss (3).

Various animal models have been developed to study cancer-associated wasting. A common practice in the models is for the primary tumor to remain as a pathogen for the duration of the study to induce wasting (2, 4). Studies using such models have generated useful findings indicating the underlying biological mechanisms of the nature and complexity of cachexia. However, the presence of the primary tumor and severity of the wasting (e.g. significant weight loss and metabolic disturbance) may make such models less useful for studies aimed at early treatment and prevention of wasting.

In our laboratory, we study the effects of dietary factors on malignant spread using a spontaneous metastasis model in which the primary tumor is surgically removed. This allows us to assess treatment effects on metastatic development and growth without the influence of excessive nutrient deprivation and marked reduction in body weight due to the rapidly growing primary tumor (5, 6). In preliminary assessments of bone specimens from studies in which curcumin enhanced metastatic growth of Lewis lung carcinoma (LLC) (5, 7), we found undesirable changes in trabecular microarchitecture in LLC-bearing mice (unpublished data). This observation suggested that bone loss might be an early pathophysiological change in wasting associated with metastatic progression.

Obesity is a leading risk factor for cancer (8), and excessive body fat mass is detrimental to skeletal health (9). Adipose tissue is considered an endocrine organ that produces inflammatory cytokines, e.g. plasminogen activator inhibitor-1 (PAI1), that contribute to obesity and obesity-related cancer. We found that a high-fat diet enhances, whereas PAI1 deficiency attenuates spontaneous metastasis of LLC in mice (10). Thus, we hypothesized that metastasis of LLC is accompanied with bone loss. We further hypothesized that a high-fat diet enhances metastasis-associated bone loss and that PAI1 deficiency reduces this loss. To test these hypotheses, we performed micro-computed tomographic analysis of femurs and vertebrae collected from our previously completed study showing that a high-fat diet and PAI1 deficiency affect spontaneous metastasis (10).

Materials and Methods

Animals and diets. Four to five-week-old male PAI1-deficient mice (Pai1−/−, B6.129S2-Serpine1tm1Mlg/J) with a C57BL/6J background and C57BL/6J wild-type mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were fed AIN93G diet (11) modified with 16% of energy from corn oil, or AIN93G diet with 45% of energy from corn oil (hereafter referred to as the high-fat diet) (10). Mice were housed in a pathogen-free room with a 12:12-hour light-dark cycle and maintained at 22±1°C. Mice were weighed weekly; they had free access to their diets and deionized water. All diets were powdered; they were stored at −20°C until being provided to mice.

LLC cells. LLC cell line, a variant that metastasizes specifically to lungs (12), was obtained from Dr. Pnina Brodt, McGill University, Montreal, Quebec, Canada. The cells were cultured with RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum and maintained in a humidified atmosphere of 5% CO2 in air at 37°C. Cells used for animal studies were in vivo-selected once (13). The cells were monitored for phenotype (by microscopic examination of cell morphology), proliferation properties (by growth curve analysis) and metastatic capability (by injecting cells subcutaneously into mice and examining metastatic formation in lungs). Cells were free of mycoplasma based on Hoechst DNA staining and direct culture tests performed by American Type Cell Collection (Manassas, VA, USA). These assessments showed that cell identity and metastatic behavior were similar to those of original stocks from the institution providing the cell line.

Experimental design. The study (YAN019) was approved by the Animal Care and Use Committee of the U.S. Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center. The procedures followed the guidelines of the National Institute of Health for the care and use of laboratory animals (14). The experimental regimen was reported previously (10). Briefly, Pai1−/− and wild-type mice were fed their respective AIN93G or high-fat diet for 7 weeks before each mouse was subcutaneously injected in the lower dorsal region with 2.5×105 viable LLC cells. The resulting primary tumor was removed surgically 12 days later when it was approximately 1 cm in diameter. Following removal, the mice were maintained on their respective diets for an additional 10 days. Thirteen wild-type mice that were fed the AIN93G diet were not injected with LLC cells; they were used as controls for determining bone structural changes in AIN93G-fed LLC-bearing wild-type mice. At termination, mice were euthanized with an intraperitoneal injection of a mixture of ketamine and xylazine, and their lungs were removed for determination of the extent of pulmonary metastasis (10). The right femur and vertebral column (from 10th or 11th thoracic vertebra to the sacrum) was collected from each mouse and stored in phosphate-buffered saline for micro-computed tomographic analysis of trabecular and cortical bone. Plasma was collected for quantifying osteocalcin and tartrate-resistant acid phosphatase 5b (TRAP5b).

Bone evaluation. Femurs were cleaned with cheesecloth for physical measurements before micro-computed tomographic analysis. Femoral length along the proximal distal direction and mid-shaft widths in both medial-lateral and anterior-posterior axes were measured using an electronic digital caliper (Fred V. Fowler Company, Newton, MA, USA). Femurs and lumbar vertebral bodies were evaluated for trabecular and cortical bone structural properties using high-resolution (12-μm slice increment) micro-computed tomography (μCT-40; Scanco Medical, Basserdorf, Switzerland) with an x-ray source power of 55 keV and 145 μA, and integration time of 300 ms. A fixed threshold of 275 was used to delineate mineralized bone from soft tissue and marrow phase. In distal femurs, trabecular bone was evaluated in 125 slices (1.5 mm) of the metaphysis proximal to the distal growth plate, and cortical bone was evaluated in 100 slices (1.2 mm) at mid-shaft of the femur. In vertebral bodies, trabecular and cortical bone were analyzed along the entire cranial-caudal axis of the fourth lumbar vertebra. The evaluation followed guidelines for assessment of bone microstructure in rodents using micro-computed tomography (15).

For trabecular bone, total volume (TV, mm3), bone volume (BV, mm3), bone volume fraction (percentage ratio of the segmented BV to the TV of the region evaluated, BV/TV, %), connectivity density (a degree of connectivity of trabeculae normalized by TV, Conn.D, 1/mm3), structure model index (SMI; an indicator of the plate- and rod-like geometry of trabecular structure), trabecular number (the average number of trabeculae per unit length, Tb.N, 1/mm), trabecular thickness (mean thickness of trabeculae, Tb.Th, mm), trabecular separation (mean distance between trabeculae, Tb.Sp, mm) and bone mineral density (BMD; mg hydroyxapatite/cm3) were measured in distal femurs and vertebral bodies. For cortical bone, total cross-sectional area inside the periosteal envelope (Tt.Ar, mm2), cortical bone area (Ct.Ar, mm2), cortical area fraction (Ct.Ar/T.Ar, %) and average cortical thickness (Ct.Th, mm) were computed for the femoral mid-shaft and vertebral bodies.

Quantification of plasma osteocalcin and TRAP5b. Sandwich enzyme-linked immunosorbent assay kits were used for determination of plasma concentrations of osteocalcin (Biomedical Technology, Stoughton, MA, USA) and TRAP5b (Immunodiagnostic Systems, Scottsdale, AZ, USA) following protocols provided by the manufacturers. Samples were read within the linear range of the assay, and the accuracy of the analysis was confirmed by the controls provided in each assay kit.

Figure 1.
Figure 1.

Trabecular microstructural changes in femurs: a: Bone volume fraction (BV/TV); b: connectivity density (Conn.D); c: structure model index (SMI); d: trabecular number (Tb.N); e: trabecular thickness (Tb.Th); f: trabecular separation (Tb.Sp). Two-way ANOVA was performed to compare differences among the groups of Lewis lung carcinoma-bearing (LLC) mice; a priori contrasts were performed to compare differences between AIN93G-fed (AIN) wild-type (WT) non-tumor-bearing controls (Ctl) and AIN93G-fed wild-type mice with LLC (AIN WT), *p<0.05 compared to AIN WT. Values are means±SEM (n=13-14 per group). Pai1−/−: Pai1−/− mice; HF: high-fat diet.

Statistical analyses. The effects of diet (AIN93G or high-fat), genotype (Pai1−/− or wild-type) and their interaction were tested using two-way analysis of variance (ANOVA). If a significant interaction between diet and genotype occurred, Tukey contrasts were performed to compare the four treatment groups. To examine the effect of LLC on bone structural changes and plasma concentrations of osteocalcin and TRAP5b, a priori contrasts were used to test for differences in AIN93G-fed wild-type mice with and without LLC. All data are presented as means±standard error of the mean (SEM). Differences with a p-value of 0.05 or less were considered statistically significant. All analyses were performed using SAS software (version 9.3; SAS Institute, Cary, NC, USA).

Results

As reported previously (10), subcutaneous injection of LLC cells resulted in pulmonary metastasis; consumption of a high-fat diet enhanced LLC lung metastasis, and PAI1 deficiency reduced the metastasis.

Physical measurement of bone. There were no significant differences in femur length, medial-lateral axis width and anterior-posterior axis width between non-tumor-bearing and LLC-bearing mice fed the AIN93G diet (data not shown). There were also no differences in these variables between LLC-bearing wild-type and Pai1−/− mice fed the AIN93G or the high-fat diet (data not shown).

Figure 2.
Figure 2.

Trabecular microstructural changes in vertebrae: a: Bone volume fraction (BV/TV); b: connectivity density (Conn.D); c: structure model index (SMI); d: trabecular number (Tb.N); e: trabecular thickness (Tb.Th); f: trabecular separation (Tb.Sp). Two-way ANOVA was performed to compare differences among the groups of Lewis lung carcinoma-bearing (LLC) mice; a priori contrasts were performed to compare differences between AIN93G-fed (AIN) wild-type (WT) non-tumor-bearing controls (Ctl) and AIN93G-fed wild-type mice with LLC (AIN WT), *p<0.05 compared to AIN WT. Values are means±SEM (n=13-14 per group). Pai1−/−: Pai1−/− mice; HF: high-fat diet.

Micro-computed tomographic measurement of trabecular bone. The presence of LLC metastases in lungs adversely affected the three-dimensional microstructure of trabecular bone. Compared to non-tumor-bearing mice, LLC metastasis in mice reduced BV/TV by 22% (p<0.05, Figure 1a), Conn.D by 28% (p<0.05, Figure 1b), Tb.N by 8% (p<0.05, Figure 1d) and Tb.Th by 9% (p<0.05, Figure 1e) and increased Tb.Sp by 10% (p<0.05, Figure 1f) in femurs. Similarly, LLC metastasis reduced BV/TV by 21% (p<0.05, Figure 2a), Tb.N by 4% (p<0.05, Figure 2d) and Tb.Th by 13% (p<0.05, Figure 2e) and increased SMI by 69% (p<0.05, Figure 2c) and Tb.Sp by 7% (p<0.05, Figure 2f) in the lumbar vertebra 4. Metastasis of LLC reduced BMD by 17% in femurs (p<0.05, Figure 3a) and 15% in vertebrae (p<0.05, Figure 3b).

Consumption of the high-fat diet enhanced LLC-induced trabecular microstructural changes. The high-fat diet compared to the AIN93G diet reduced BV/TV by 18% (p<0.01, Figure 1a), Conn.D by 30% (p<0.01, Figure 1b), Tb.N by 8% (p<0.01, Figure 1d) and increased SMI by 8% (p<0.05, Figure 1c) and Tb.Sp by 10% (p<0.01, Figure 1f) in femurs. The high-fat diet compared to the AIN93G diet reduced BV/TV by 9% (p<0.05, Figure 2a) and increased SMI by19% (p<0.01, Figure 2c) in vertebrae. The high-fat diet compared to the AIN93G diet reduced BMD by 14% in femurs (p<0.01, Figure 3a), and it reduced BMD by 12% in vertebrae of wild-type mice (p<0.05) but not Pai1−/− mice (Figure 3b).

Figure 3.
Figure 3.

Bone mineral density (BMD) of femurs and vertebrae. Two-way ANOVA and Tukey contrasts were performed to compare differences among the groups of Lewis lung carcinoma-bearing (LLC) mice; a priori contrasts were performed to compare differences between AIN93G-fed (AIN) wild-type (WT) non-tumor-bearing controls (Ctl) and AIN93G-fed wild-type mice with LLC (AIN WT); *p<0.05 compared to AIN WT. Values (means±SEM; n=13-14 per group) with different superscripts are significantly different at p≤0.05 for LLC-bearing mice. Pai1−/−: Pai1−/− mice; HF: high-fat diet.

Compared to the wild-type mice, Pai1−/− mice exhibited fewer LLC-induced adverse trabecular changes, as evidenced by an increase in BV/TV by 19% (p<0.05, Figure 1a), Conn.D by 25% (p<0.05, Figure 1b) and Tb.N by 5% (p<0.05, Figure 1d) and a reduction in SMI by 10% (p<0.01, Figure 1c) in femurs. Deficiency in PAI1 did not affect LLC-induced trabecular microstructural changes in vertebrae (Figure 2), nor did it affect BMD in either femurs or vertebrae (Figure 3).

Micro-computed tomographic measurement of cortical bone. Compared to non-tumor-bearing mice, LLC-bearing mice exhibited decreases of 4% (p<0.05, Figure 4a) and 8% (p<0.01, Figure 4c) in Ct.Ar/Tt.Ar and 5% (p<0.05, Figure 4b) and 9% (p<0.01, Figure 4d) in Ct.Th of the mid-shaft of femurs and the vertebra cortical bone, respectively. Neither the high-fat diet nor PAI1 deficiency affected LLC-induced cortical structural changes in femurs and vertebrae compared to their respective controls (Figure 4).

Plasma concentrations of osteocalcin and TRAP5b. The presence of LLC metastases in lungs reduced plasma concentrations of osteocalcin by 24% (p<0.05, Figure 5a) and increased plasma TRAP5b by 80% (p<0.01, Figure 5b). The high-fat diet reduced plasma concentrations of osteocalcin by 33% (p<0.01, Figure 5a) compared to the AIN93G diet; it increased TRAP5b by 67% (p<0.05) in wild-type mice, but not in Pai1−/− mice (Figure 5b). Deficiency in PAI1 did not affect plasma concentrations of osteocalcin (Figure 5a). Deficiency in PAI1 also did not affect plasma concentrations of TRAP5b in AIN93G-fed mice, however, it completely prevented the high-fat diet-induced increase in TRAP5b (p<0.05, Figure 5b).

Discussion

The present study demonstrated that the presence of LLC metastases in lungs adversely affected trabecular and cortical structure and reduced bone mineral density in mice. In addition, the study showed that a high-fat diet enhanced LLC-mediated detrimental effects on bone. In contrast, PAI1 deficiency attenuated LLC-induced detrimental changes in trabecular bone, which indicates that PAI1 may play a role in LLC-mediated skeletal deterioration.

The detrimental changes in trabecular and cortical bone in mice with LLC metastases were accompanied by a decrease in plasma concentration of osteocalcin and an increase in TRAP5b. Bone remodeling is an adaptive mechanism that controls bone mass and microachitecture throughout life. The remodeling occurs through the coordinated, coupled activity of bone formation and resorption. Osteocalcin is a marker of bone formation (16, 17). For example, changes in serum concentrations of osteocalcin reflect the status of bone formation in post-menopausal osteoporosis (18, 19). Serum concentration of osteocalcin during the growth period has been positively correlated to the appositional rate, the rate of longitudinal bone growth, the rate of production of chondrocytes in growth plate and the thickness of the growth plate in animals (20). TRAP5b is a marker of bone resorption (21, 22). For example, serum TRAP5b activity was significantly elevated in patients with osteoporosis and inversely correlated with bone mineral density (23). The concentration of TRAP5b in serum has also been shown to correlate with osteoclast surface and osteoclast number in patients with metabolic bone diseases (24). Our results suggest that LLC metastasis may uncouple bone formation and resorption; this may be the reason for the bone loss indicated by structural changes in LLC-bearing mice.

The metastatic aggressiveness of LLC is accompanied by significant increases in plasma concentrations of inflammatory cytokines [PAI1, monocyte chemoattractant protein-1 (MCP1), tumor necrosis factor-α (TNFα)], proteases [urokinase, matrix metalloproteinase-9 (MMP9)] and angiogenic factors [vascular endothelial growth factor (VEGF), tissue inhibitor of metalloproteinase-1 (TIMP1)](10, 25). Elevated amounts of these inflammatory cytokines, proteases and angiogenic factors are associated with bone loss. In vitro studies showed that the plasminogen activator system, including urokinase and PAI1, is involved in bone metabolism (26). Bone loss is reduced by PAI1 deficiency in estrogen-deficient (27) and diabetic mice (28). Similarly, MCP1 deficiency has been found to increase bone mass in femurs and reduce the numbers and activity of osteoclasts in vitro (29). Osteoclastogenesis and osteoclast activation are promoted by TNFα (30, 31). Overexpression of MMP9 is associated with increased bone resorption (32). An inhibitor of MMP9, TIMP1, might play a dual role in bone through stimulating bone resorption at low concentrations and reducing it at higher concentrations (33). Promotion of osteoclastogenesis (34) and osteoblast differentiation (35) by VEGF has been reported but depends on the models used. While the exact mechanisms by which LLC metastasis reduces bone mass remain to be elucidated, our results suggest that up-regulation of production of inflammatory cytokines, proteases and angiogenic factors may contribute to bone loss.

Figure 4.
Figure 4.

Changes in cortical bone of femurs and vertebrae: a, c: Cortical area fraction (Ct.Ar/Tt.Ar); b, d: cortical thickness (Ct.Th). Two-way ANOVA was performed to compare differences among the groups of Lewis lung carcinoma-bearing (LLC) mice; a priori contrasts were performed to compare differences between AIN93G-fed (AIN) wild-type (WT) non-tumor-bearing controls (Ctl) and AIN93G-fed wild-type mice with LLC (AIN WT); *p<0.05 compared to AIN WT. Values are means±SEM (n=13-14 per group). Pai1−/−: Pai1−/− mice; HF: high-fat diet.

LLC metastasizes spontaneously to lungs when these cells are injected subcutaneously into mice (6, 12). Our results clearly demonstrate that bone loss occurs in mice with the development and progression of LLC metastases. Furthermore, it indicates that the spontaneous metastasis model, which avoids drastic nutrient deprivation and weight loss by surgical removal of the rapidly growing primary tumor, is advantageous in assessing metastasis-related bone loss over cachexia models (2, 4), in which the primary tumor remains intact as a pathogen to induce wasting syndrome and bone loss may be a result of multiple detrimental effects in addition to metastasis. Thus, this model is useful for studying not only malignant spread but also simultaneous bone loss. It is of importance because skeletal deterioration may have critical long-term health consequences for patients with cancer.

Figure 5.
Figure 5.

Plasma concentrations of osteocalcin and tartrate-resistant acid phosphatase 5b (TRAP5b). Two-way ANOVA and Tukey contrasts were performed to compare differences among the groups of Lewis lung carcinoma-bearing (LLC) mice; a priori contrasts were performed to compare differences between AIN93G-fed (AIN) wild-type (WT) non-tumor-bearing controls (Ctl) and AIN93G-fed wild-type mice with LLC (AIN WT); *p<0.05 compared to AIN WT. Values (means±SEM; n=10 per group) with different superscripts are significantly different at p≤0.05 for LLC-bearing mice. Pai1−/−: Pai1−/− mice; HF: high-fat diet.

The significant deterioration in trabecular microstructure and reduction in bone mineral density in LLC-bearing mice fed the high-fat diet was accompanied by a significant decrease in the concentration of osteocalcin and a significant increase in TRAP5b in plasma. This finding indicates that the enhancement of bone loss by a high-fat diet might be through the uncoupling of bone formation and resorption. This is consistent with reports that obesity induced by a high-fat diet is detrimental to bone health (36, 37).

The high-fat diet significantly increased body weights in the present study (10). This increase was the result of an increase in body fat mass but not in absolute lean mass weight (10). Adipose tissue is considered an endocrine organ that produces adipokines that contribute to obesity and obesity-related cancer. Consumption of a high-fat diet enhances malignant spread and metastatic growth of LLC in lungs (10, 25) and also significantly increases plasma concentrations of adipokines (PAI1, MCP1, TNFα, leptin), proteases (urokinase, MMP9) and angiogenic factors (VEGF, TIMP9) (10, 25). As discussed above, an increase of these adipokines (27-31), proteases (26, 32) and angiogenic factors (33, 34) adversely affects bone metabolism. Thus, their elevation likely contributes to the high-fat diet-enhanced bone loss in metastasis-related wasting in LLC-bearing mice.

The serine protease inhibitor PAI1 promotes primary tumor growth (38) and metastasis (10, 39). The attenuation of LLC-induced detrimental changes in trabecular bone by PAI1 deficiency indicates that PAI1 actively contributes to LLC-mediated bone loss. It has been found that PAI1 deficiency prevents bone loss in estrogen-deficient mice (27) and streptozotocin-induced diabetic mice (28). A reversal in the decline of alkaline phosphatase-positive cells was found in a study where PAI1 deficiency attenuated diabetes-impaired bone health in mice (40). In the present study, PAI1 deficiency significantly reduced plasma concentrations of TRAP5b in LLC-bearing mice fed the high-fat diet. This finding is consistent with reports that PAI1 deficiency protects against experimentally induced bone loss (27, 28, 40). It indicates that PAI1 may interrupt bone remodeling by promoting bone resorption and thus contributes to LLC-mediated bone deterioration.

PAI1 and urokinase are recommended as prognostic markers in breast cancer (41). An inhibitor of PAI1 (42) and PAI1-specific RNA aptamers (43) have been developed for pre-clinical studies of their potential anticancer application (42). Considering that PAI1 exacerbates bone loss in metastasis, it may be suitable to include skeletal health as an end-point measurement in studies that assess PAI1 inhibition in cancer prevention and treatment.

Neither PAI1 deficiency nor the high-fat diet affected cortical bone properties in the present study. The finding that trabecular rather than cortical bone is affected by PAI1 deficiency or the high-fat diet is not surprising. Trabecular bone is a complex network made up of two types of trabeculae: rods and plates (44). It is the most metabolically-active and major site of bone remodeling under physiological conditions, thus it is more responsive to experimental treatments than cortical bone because of its larger surface to volume ratio (45). On the other hand, cortical bone, because of its function in supporting the whole body and protecting organs, is much denser and stiffer in structure than trabecular bone. Thus, it takes longer to adapt to macro-remodeling or show changes in geometry (45), which could not be achieved in the present study.

In summary, the present study found detrimental changes in trabecular and cortical bone in mice with LLC metastases in lungs. These pathophysiological changes indicate that bone wasting is associated with the development and progression of metastasis. Consumption of a high-fat diet enhanced LLC-mediated bone loss, and PAI1 deficiency attenuated this loss. This indicates that the high-fat diet and PAI1 directly contribute to bone loss in metastasis-associated wasting. Furthermore, it suggests that the spontaneous metastasis model used in the present study may be useful for studying not only malignant spread but also bone loss of metastasis-related wasting simultaneously.

Acknowledgements

The Authors greatly acknowledge the assistance of the following staff of Grand Forks Human Nutrition Research Center: Lana DeMars and Kay Keehr for technical support, James Linlauf for preparing experimental diets and vivarium staff for providing high quality animal care. Funding for this work was provided by the U.S. Department of Agriculture, ARS, research project 5450-51000-045-00D.

Mention of trade names or commercial products in this article is solely for providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

Footnotes

  • Conflicts of Interest

    The Authors declare no competing financial interest in regard to this study.

  • Received April 11, 2015.
  • Revision received May 6, 2015.
  • Accepted May 7, 2015.

References

  1. ↵
    1. Ellis KJ,
    2. Yasumura S,
    3. Morgan WD
    1. Preston T,
    2. Fearon KCH,
    3. Robertson I,
    4. East BW,
    5. Calman KC
    : Tissue loss during severe wasting in lung cancer patients. In: In Vivo Body Composition Studies. Ellis KJ, Yasumura S, Morgan WD (ed.). London, Institute of Physical Sciences in Medicine, pp. 60-69, 1987.
  2. ↵
    1. Choi E,
    2. Carruthers K,
    3. Zhang L,
    4. Thomas N,
    5. Battaglino RA,
    6. Morse LR,
    7. Widrick JJ
    : Concurrent muscle and bone deterioration in a murine model of cancer cachexia. Physiol Rep 1: e00144, 2013.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Kandarian S
    : The molecular basis of skeletal muscle atrophy–parallels with osteoporotic signaling. J Musculoskelet Neuronal Interact 8: 340-341, 2008.
    OpenUrlPubMed
  4. ↵
    1. Nicholson JR,
    2. Kohler G,
    3. Schaerer F,
    4. Senn C,
    5. Weyermann P,
    6. Hofbauer KG
    : Peripheral administration of a melanocortin 4-receptor inverse agonist prevents loss of lean body mass in tumor-bearing mice. J Pharmacol Exp Ther 317: 771-777, 2006.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Yan L
    : Dietary supplementation with curcumin enhances metastatic growth of Lewis lung carcinoma in mice. Int J Cancer 132: 269-275, 2013.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Yan L,
    2. DeMars LC
    : Dietary supplementation with methylseleninic acid, but not selenomethionine, reduces spontaneous metastasis of Lewis lung carcinoma in mice. Int J Cancer 131: 1260-1266, 2012.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Yan L,
    2. Yee JA,
    3. Cao J
    : Curcumin reduces trabecular and cortical bone in naive and Lewis lung carcinoma-bearing mice. Anticancer Res 33: 3153-3161, 2013.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Bianchini F,
    2. Kaaks R,
    3. Vainio H
    : Overweight, obesity, and cancer risk. Lancet Oncol 3: 565-574, 2002.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Zhao LJ,
    2. Liu YJ,
    3. Liu PY,
    4. Hamilton J,
    5. Recker RR,
    6. Deng HW
    : Relationship of obesity with osteoporosis. J Clin Endocrinol Metab 92: 1640-1646, 2007.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Yan L,
    2. DeMars LC
    : Effects of a high-fat diet on spontaneous metastasis of Lewis lung carcinoma in plasminogen activator inhibitor-1 deficient and wild-type mice. PLoS ONE 9: e110869, 2014.
    OpenUrlPubMed
  11. ↵
    1. Reeves PG,
    2. Nielsen FH,
    3. Fahey GCJ
    : AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 123: 1939-1951, 1993.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Brodt P
    : Characterization of two highly metastatic variants of Lewis lung carcinoma with different organ specificities. Cancer Res 46: 2442-2448, 1986.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Yan L,
    2. Demars LC
    : Effects of dietary fat on spontaneous metastasis of Lewis lung carcinoma in mice. Clin Exp Metastasis 27: 581-590, 2010.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Institute for Laboratory Animal Research
    : Guide for the Care and Use of Laboratory Animals (8th ed). Washington, D.C., National Academies Press, 2011.
  15. ↵
    1. Bouxsein ML,
    2. Boyd SK,
    3. Christiansen BA,
    4. Guldberg RE,
    5. Jepsen KJ,
    6. Muller R
    : Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone Miner Res 25: 1468-1486, 2010.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Weinreb M,
    2. Shinar D,
    3. Rodan GA
    . Different pattern of alkaline phosphatase, osteopontin, and osteocalcin expression in developing rat bone visualized by in situ hybridization. J Bone Miner Res 5: 831-842, 1990.
    OpenUrlPubMed
  17. ↵
    1. Boivin G,
    2. Morel G,
    3. Lian JB,
    4. Anthoine-Terrier C,
    5. Dubois PM,
    6. Meunier PJ
    : Localization of endogenous osteocalcin in neonatal rat bone and its absence in articular cartilage: effect of warfarin treatment. Virchows Arch A Pathol Anat Histopathol 417: 505-512, 1990.
    OpenUrlPubMed
  18. ↵
    1. Brown JP,
    2. Delmas PD,
    3. Malaval L,
    4. Edouard C,
    5. Chapuy MC,
    6. Meunier PJ
    : Serum bone GLA-protein: a specific marker for bone formation in postmenopausal osteoporosis. Lancet 1: 1091109-3, 1984.
    OpenUrl
  19. ↵
    1. Brown JP,
    2. Delmas PD,
    3. Arlot M,
    4. Meunier PJ
    : Active bone turnover of the cortico-endosteal envelope in postmenopausal osteoporosis. J Clin Endocrinol Metab 64: 954-959, 1987.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Pastoureau P,
    2. Meunier PJ,
    3. Delmas PD
    : Serum osteocalcin (bone GLA-protein), an index of bone growth in lambs. Comparison with age-related histomorphometric changes. Bone 12: 143-149, 1991.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Halleen JM,
    2. Alatalo SL,
    3. Suominen H,
    4. Cheng S,
    5. Janckila AJ,
    6. Vaananen HK
    : Tartrate-resistant acid phosphatase 5b: a novel serum marker of bone resorption. J Bone Miner Res 15: 1337-1345, 2000.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Kirstein B,
    2. Chambers TJ,
    3. Fuller K
    : Secretion of tartrate-resistant acid phosphatase by osteoclasts correlates with resorptive behavior. J Cell Biochem 98: 1085-1094, 2006.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Halleen JM,
    2. Ylipahkala H,
    3. Alatalo SL,
    4. Janckila AJ,
    5. Heikkinen JE,
    6. Suominen H,
    7. Cheng S,
    8. Vaananen HK
    : Serum tartrate-resistant acid phosphatase 5b, but not 5a, correlates with other markers of bone turnover and bone mineral density. Calcif Tissue Int 71: 20-25, 2002.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Ballanti P,
    2. Minisola S,
    3. Pacitti MT,
    4. Scarnecchia L,
    5. Rosso R,
    6. Mazzuoli GF,
    7. Bonucci E
    : Tartrate-resistant acid phosphate activity as osteoclastic marker: sensitivity of cytochemical assessment and serum assay in comparison with standardized osteoclast histomorphometry. Osteoporos Int 7: 39-43, 1997.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Yan L,
    2. Combs GFJ
    : Consumption of a high-fat diet abrogates inhibitory effects of methylseleninic acid on spontaneous metastasis of Lewis lung carcinoma in mice. Carcinogenesis 35: 2308-2313, 2014.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Daci E,
    2. Udagawa N,
    3. Martin TJ,
    4. Bouillon R,
    5. Carmeliet G
    : The role of the plasminogen system in bone resorption in vitro. J Bone Miner Metab 14: 946-952, 1999.
    OpenUrl
  27. ↵
    1. Daci E,
    2. Verstuyf A,
    3. Moermans K,
    4. Bouillon R,
    5. Carmeliet G
    : Mice lacking the plasminogen activator inhibitor 1 are protected from trabecular bone loss induced by estrogen deficiency. J Bone Miner Res 15: 1510-6151, 2000.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Kawao N,
    2. Tamura Y,
    3. Okumoto K,
    4. Yano M,
    5. Okada K,
    6. Matsuo O,
    7. Kaji H
    : Plasminogen plays a crucial role in bone repair. J Bone Miner Res 28: 1561-1574, 2013.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Sul OJ,
    2. Ke K,
    3. Kim WK,
    4. Kim SH,
    5. Lee SC,
    6. Kim HJ,
    7. Suh JH,
    8. Choi HS
    : Absence of MCP1 leads to elevated bone mass via impaired actin ring formation. J Cell Physiol 227: 1619-27, 2012.
    OpenUrlPubMed
  30. ↵
    1. Fuller K,
    2. Murphy C,
    3. Kirstein B,
    4. Fox SW,
    5. Chambers TJ
    : TNFalpha potently activates osteoclasts, through a direct action independent of and strongly synergistic with RANKL. Endocrinology 143: 1108-1118, 2002.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Lam J,
    2. Takeshita S,
    3. Barker JE,
    4. Kanagawa O,
    5. Ross FP,
    6. Teitelbaum SL
    : TNF-alpha induces osteoclastogenesis by direct stimulation of macrophages exposed to permissive levels of RANK ligand. J Clin Invest 106: 1481-1488, 2000.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Grassi F,
    2. Cristino S,
    3. Toneguzzi S,
    4. Piacentini A,
    5. Facchini A,
    6. Lisignoli G
    : CXCL12 chemokine up-regulates bone resorption and MMP-9 release by human osteoclasts: CXCL12 levels are increased in synovial and bone tissue of rheumatoid arthritis patients. J Cell Physiol 199: 244-251, 2004.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Bildt MM,
    2. Bloemen M,
    3. Kuijpers-Jagtman AM,
    4. Von den Hoff JW
    : Matrix metalloproteinases and tissue inhibitors of metalloproteinases in gingival crevicular fluid during orthodontic tooth movement. Eur J Orthod 31: 529-535, 2009.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Peyruchaud O,
    2. Serre CM,
    3. NicAmhlaoibh R,
    4. Fournier P,
    5. Clezardin P
    : Angiostatin inhibits bone metastasis formation in nude mice through a direct anti-osteoclastic activity. J Biol Chem 278: 45826-45832, 2003.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. Kitagawa Y,
    2. Dai J,
    3. Zhang J,
    4. Keller JM,
    5. Nor J,
    6. Yao Z,
    7. Keller ET
    : Vascular endothelial growth factor contributes to prostate cancer-mediated osteoblastic activity. Cancer Res 65: 10921-10929, 2005.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Cao JJ,
    2. Gregoire BR,
    3. Gao H
    : High-fat diet decreases cancellous bone mass but has no effect on cortical bone mass in the tibia in mice. Bone 44: 1097-1104, 2009.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Patsch JM,
    2. Kiefer FW,
    3. Varga P,
    4. Pail P,
    5. Rauner M,
    6. Stupphann D,
    7. Resch H,
    8. Moser D,
    9. Zysset PK,
    10. Stulnig TM,
    11. Pietschmann P
    : Increased bone resorption and impaired bone microarchitecture in short-term and extended high-fat diet-induced obesity. Metabolism 60: 243-249, 2011.
    OpenUrlCrossRefPubMed
  38. ↵
    1. McMahon GA,
    2. Petitclerc E,
    3. Stefansson S,
    4. Smith E,
    5. Wong MK,
    6. Westrick RJ,
    7. Ginsburg D,
    8. Brooks PC,
    9. Lawrence DA
    : Plasminogen activator inhibitor-1 regulates tumor growth and angiogenesis. J Biol Chem 276: 33964-33968, 2001.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Tsuchiya H,
    2. Sunayama C,
    3. Okada G,
    4. Matsuda E,
    5. Tomita K,
    6. Binder BR
    . Plasminogen activator inhibitor-1 accelerates lung metastasis formation of human fibrosarcoma cells. Anticancer Res 17: 313-316, 1997.
    OpenUrlPubMed
  40. ↵
    1. Mao L,
    2. Kawao N,
    3. Tamura Y,
    4. Okumoto K,
    5. Okada K,
    6. Yano M,
    7. Matsuo,
    8. Kaji H
    : Plasminogen activator inhibitor-1 is involved in impaired bone repair associated with diabetes in female mice. PLoS ONE 9: e92686, 2014.
    OpenUrlCrossRefPubMed
  41. ↵
    1. Harris L,
    2. Fritsche H,
    3. Mennel R,
    4. Norton L,
    5. Ravdin P,
    6. Taube S,
    7. Somerfield MR,
    8. Hayes DF,
    9. Bast RC Jr..
    : American Society of Clinical Oncology 2007 update of Recommendations for the Use of Tumor Markers in Breast Cancer. J Clin Oncol 25: 5287-5312, 2007.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Leik CE,
    2. Su EJ,
    3. Nambi P,
    4. Crandall DL,
    5. Lawrence DA
    . Effect of pharmacologic plasminogen activator inhibitor-1 inhibition on cell motility and tumor angiogenesis. J Thromb Haemost 4: 2710-2715, 2006.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Blake CM,
    2. Sullenger BA,
    3. Lawrence DA,
    4. Fortenberry YM
    : Antimetastatic potential of PAI1-specific RNA aptamers. Oligonucleotides 19: 117-128, 2009.
    OpenUrlPubMed
  44. ↵
    1. Parfitt AM,
    2. Mathews CH,
    3. Villanueva AR,
    4. Kleerekoper M,
    5. Frame B,
    6. Rao DS
    : Relationships between surface, volume, and thickness of iliac trabecular bone in aging and in osteoporosis. Implications for the microanatomic and cellular mechanisms of bone loss. J Clin Invest 72: 1396-409, 1983.
    OpenUrlCrossRefPubMed
  45. ↵
    1. Marcus R,
    2. Feldman D,
    3. Nelson DA,
    4. Rosen CJ
    1. Morgan EF,
    2. Barnes GL,
    3. Einhorn TA
    : The bone organ system: form and function. In: Osteoporosis, Marcus R, Feldman D, Nelson DA, Rosen CJ (eds.) Third Edition. Burlington, MA, Elsevier Academic Press, pp. 3-25, 2008.
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High-fat Diet Enhances and Plasminogen Activator Inhibitor-1 Deficiency Attenuates Bone Loss in Mice with Lewis Lung Carcinoma
LIN YAN, FORREST H. NIELSEN, SNEHA SUNDARAM, JAY CAO
Anticancer Research Jul 2015, 35 (7) 3839-3847;
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