Bone Integrity Affects the Natural History of Prostate Cancer
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By Stephen B. Strum, M.D.

Reprinted from PCRI Insights January, 1999 vol. 2, no. 1

Much of this issue of Insights is devoted to an in-depth discussion of bone integrity. Why so much emphasis on bone? Every so often I come across a topic in PC that rivets my attention. Bone integrity and the factors that relate to growth of PC in bone, why PC spreads to bone, the nature of bone pain from PC and the issue of prevention of bone metastases by changing the micro-environment are important pieces in the puzzle of what we need to solve about PC. Bear with me and read through this issue.

In the last issue of Insights, we discussed three concepts relating to bone integrity:

We used the analogy of the bone as a bank account with bone density being the balance, and formation and resorption being deposits and withdrawals, respectively.

When excessive bone resorption persists, a loss in bone mass results. If unchecked, it eventually leads to osteoporosis. Osteoporosis is a critical health issue in the United States; 1 of 2 women and 1 of 8 men older than 50 years of age are expected to have bone fractures. The cost of osteoporotic related illness in the United States is $38 million dollars a day or $14 billion dollars each year. The ratio of bone fractures for men becomes significantly higher when men are subjected to castration since the abrupt decrease in androgens is analogous to what women experience at menopause at the time of abrupt decrease in estrogens. Male menopause is induced by androgen deprivation therapy (ADT), be it from surgical castration, the use of an LHRH agonist like Lupron® or Zoladex®, or sequential androgen blockade (SAB) using an anti-androgen (Casodex®, Eulexin®, or Nilandron®) with a 5 alpha reductase inhibitor (Proscar®).

ADT is an abrupt event. It results in more osteoporosis and related fractures than that observed during natural male menopause. Male menopause induced by castration, from any cause, is an accelerated, compressed, and intensified menopause in contrast to natural menopause, where a gradual loss of androgens occurs over decades.

In men with prostate cancer undergoing ADT, bone resorption begins immediately. This has been documented in publications looking at the alterations of bone architecture as a result of orchiectomy1-5 as well as a side effect of LHRH agonist therapy.6-8

 

Orchiectomy Causes More Bone Loss Than LHRH Agonists

Compared to orchiectomy, there is a preferential preservation of bone mineralization and less loss of bone osteoid with the use of LHRH agonists. When orchiectomy is performed, the body reacts to the loss of testosterone by stimulating the pituitary to release LH and FSH. The high levels of FSH and LH are also associated with increased levels of ACTH (adrenocorticotrophic hormone) produced by the pituitary with consequent increased levels of cortisol. It is the increased cortisol levels that suppress the osteoblast (the cell that lays down osteoid to initiate new bone formation). These changes do not occur with LHRH agonist therapy or with treatments involving estrogens. LH and FSH production are decreased by these agents, and there is no reflex stimulation of the pituitary gland.9

 

The Osteoclast Is The Mediator of Bone Resorption

Figure 1: Osteoclasts - Mediators of Bone ResorptionThe bone architecture or matrix is essentially a lattice work of collagen fibrils that are mineralized. Bone resorption relates to the disruption of the matrix with loss of minerals and fragmentation of the collagen. Increased bone resorption results from activation of osteoclasts (cells that have the capability of destroying bone matrix by secreting acids and digestive enzymes). Androgens and estrogens stabilize or inhibit the osteoclast as well as stimulate the bone-forming cells, the osteoblasts.10 A decline or withdrawal of any of these hormones leads to excessive osteoclastic activity and resultant bone resorption. Osteoclastic resorption of bone is characterized by the development of cavities or lacunae (lakes). In the foreground of Figure 1, two osteoclasts (arrows) are shown lying in their lacunae. In contrast to the bone-eroding osteoclasts, osteoblasts are bone-forming cells. Osteoblasts are derived from precursor cells in the blood. The osteoblasts migrate to areas where bone has been eroded by osteoclasts and lay down collagen and minerals in the cavities. The bone undergoes constant remodeling with osteoclastic activity intimately coupled with osteoblastic activity. There is a close signaling between these cells, both in normal individuals and in those with bone metastases.

We are beginning to understand the roles of androgen and estrogen, as well as various growth factors or cytokines. These include transforming growth factor beta, insulin growth factor-1, parathyroid-hormone related protein, interleukin-1, and interleukin-6, prostaglandins, calcium, vitamin D and analogs of vitamin D. How these mediators interact among the tumor cell, the bone matrix, the osteoclast and the osteoblast is just now beginning to be unraveled.11-20 This is shown schematically in Figure 2.21

Figure 2: The Vicious Cycle of Tumor and Bone-Derived Growth Factors

Key Abbreviations
ADT: androgen deprivation therapy is any treatment that decreases the availability of male hormone (androgens) to the prostate cancer cell population. This can occur by decreasing Testosterone (T), by removing the testicles surgically by orchiectomy, or by the use of LHRH agonists such as Lupron®, Zoladex® or Triptorelin®. It can also be accomplished by the use of anti-androgens such as Eulexin®, Casodex® or Nilandron®, either alone or in combination with Proscar®. Other agents such as Nizoral®, DES® are also examples of ADT, with additional anti-tumor effects not mediated by androgen deprivation.
ACTH: adrenocorticotrophic hormone
ATF: amino terminal fragment (highly active part of uPA molecule)
DES: diethylstibestrol
EGF: epidermal growth factor
FSH: follicle stimulating hormone
HMW-uPA: high molecular weight uPA
IGF-1: insulin growth factor 1
IGFBPs: insulin growth factor binding proteins
IL-1: interleukin 1
IL-6: interleukin 6
IL1R and IL6R: receptors for IL-1 and IL-6
LH: luteinizing hormone
MMP-2: matrix metalloprotease 2
PDGF: platelet-derived growth factor
PTHrP: parathormone related protein
Resorption: act of removal by absorption
RH: releasing hormone
TGF-b: transforming growth factor beta
TNF-a: tumor necrosis factor-alpha
uPA: urokinase plasminogen activator

Tumor cells try to survive by producing cell products that stimulate the cell’s own growth (autocrine loops) or by elaborating proteins or enzymes that affect nearby cells (paracrine loops). For example, uPA (urokinase plasminogen activator) is a key substance made by the tumor cell that is able to self-stimulate both the tumor cell (autocrine loop) and the nearby osteoblast (paracrine loop). PTHrP, elaborated by neuroendocrine cells that make CGA (chromogranin A), is involved with uPA in similar activities.

The uPA also cleaves IGFBPs (insulin growth factor binding proteins) to release IGFs that not only stimulate osteoblast growth, (which in turn makes more IGF-1), but also allows the IGF-1 to turn on uPA production within the tumor cell (paracrine loop). Other interactions are discussed in the following scenario.

A possible scenario: Osteoblastic growth utilizes calcium and causes a drop in serum calcium stimulating osteoclastic bone resorption to lyse (dissolve) bone to maintain serum calcium. This is accompanied by an increase in parathormone (PTH) and vitamin D levels which are also trying to maintain calcium homeostasis. The osteoclastic activity releases bone-derived growth factors such as insulin growth factor-1 (IGF-1) and transforming growth factor beta (TGF-beta). These in turn stimulate the tumor cell population to grow and release PTHrP and uPA. The uPA cleaves insulin growth factor binding proteins 1 & 2 (IGF BPs 1-2) to release IGF-1 and IGF-2. The uPA and the IGFs as well as interleukin- 1 (IL-1) also stimulate the osteoblasts to produce IL-6. IL-6 stimulates activity of mature osteoclasts as well as osteoclast precursor cells which have been shown to have IL-6 receptors (IL-6R).

The tumor cell within the bone also produces TGF-b which stimulates release of PTHrP and matrix metalloprotease 2 (MMP-2), the latter of which dissolves collagen1. MMP-2 also cleaves a less active form of uPA (HMW-uPA) into a more active form (ATF) which in turn stimulates osteoblast growth. The tumor cell also has receptors for IGF-1 which in turn stimulates production of uPA, as mentioned previously.

 

What Are The Implications For Treatment?

The tumor cells survival mechanisms are elaborate. However, as these mechanisms become better understood, they give us new opportunities to block the action of cytokines, proteins and enzymes. Agouron® 3340, for example, is an investigational agent that blocks MMP-2 (as well as MMP- 3, 9 and 13).

There are other autocrine loops and paracrine loops of importance that are not shown in Figure 2 due to lack of space. PC cell lines express cytokine factors and their receptors for GM-CSF, M-CSF, SCF and G-CSF (autocrine loops).22 These factors are also commonly found in the bone marrow (paracrine loops). Perhaps sampling the marrow of high-risk patients using micrometastatic assay approaches as is being done by Impath Labs, and evaluating positive assays by incubating them with these various growth factors would give us ways to manipulate tumor growth as well as to caution us on the use of various growth factors in certain patient subsets.

Endothelin-1® (ET-1) is another PC cell product that stimulates osteoblastic growth and may also mediate the pain associated with bone metastases by virtue of its potent vasoconstrictor properties. High affinity ET-1 receptors were found on osteoblasts and ET-1 increased alkaline phosphatase activity during new bone formation in vivo. Moreover, 58% of men with advanced PC had significantly higher levels of ET-1 than the control group.23

 

The Role Of Bisphosphonates

Table 1: Bisphosphonates: Structure & Relative PotencyOverstimulation of the osteoclast cell population, from whatever cause(s), leads to net resorption of bone. Now enter the Bisphosphonates (BPs), a class of agents that fixes this problem and throws in some extras to boot. BPs all contain a P-C-P (phosphorus-carbon-phosphorus) backbone that is structurally an analogue of naturally occurring pyrophosphate P-O-P (O is an oxygen molecule rather than a carbon molecule). See Table 1.

In other words, BPs mimic pyrophosphate and bind to the hydroxyapatite crystals in the bone matrix. In this mineral-bound form, they inhibit attachment of the osteoclasts to the bone matrix and interfere with signaling to osteoclast cell precursors that normally directs them to a point of attachment on the matrix.24-25 The proposed cellular actions of BPs and the amino bisphosphonates (ABPs) which include the 2nd and 3rd generation BPs, are represented in Figure 2 by stars. BPs also act directly on the osteoclast to cause programmed cell death or apoptosis.26-27

BPs have been shown to interfere with osteoblast-mediated osteoclast activation.28 The actual mechanism of this interaction needs clarification and for this reason, we have not shown this as an area of BP blockade in Figure 2.

Recent excitement has been generated by evidence that amino bisphosphonates (ABPs) also act directly on tumor cells to cause apoptosis29-30 and also dose-dependently inhibit the adhesion of tumor cells to bone.31 A summary of cellular mechanisms of the BPs is as follows:

  • Causes apoptosis (cell death) of the
    osteoclast
  • Interferes with signaling of osteclast precursor cells attracting
    them to bone matrix
  • Interferes with osteoclast attachment to bone matrix at hydroxyapatite interface
  • Inhibits osteoblast-mediated osteoclast activation
  • Apoptosis of the tumor cell
  • Interferes with adhesion of tumor cell to bone matrix (only the Amino BPs do this).

ABPs may turn out to be one of the most critical class of drugs we can employ to prevent bone metastases or to treat established bone metastases to reduce further spread as well as to kill tumor cells. Our April 1996 paper “Bisphosphonates” discussed this potential application of BPs and also their use in reducing bone pain in metastatic PC. Please download this still current paper off our Web site at www.prostate-cancer.org.

Areas of clinical importance of BPs are shown as follows:

  • Prevention of osteoporosis
  • Decreasing fractures, compression of bone
  • Decreasing bone pain due to osteoporosis or malignancy
  • Decreasing bone metastases
  • Treatment of hypercalcemia
  • Pushing calcium into bone formation.

 

What To Do Next?

Discuss these findings with your doctor and show him the references relating to articles in this exciting field. Determine your bone status with a bone mineral density (BMD) assessment along with a first or second voided urine specimen for Pyrilinks-D®. The latter test is one of the measurements of bone breakdown that is increased with excessive bone resorption. Excessive resorption may result from ADT, PC in the bone, the use of steroids or from other factors mentioned in the first issue of Insights. Since this a critical issue in the prevention and treatment of bone metastases, a discussion of bone integrity evaluation and management is warranted.

 

Evaluation of Bone Integrity & Management

Table 3: Evaluation of Bone Mass MeasurementThe evaluation of bone integrity involves applying the principles learned in the previous pages. Bone mineral density (BMD) peaks at the age of 25 and ebbs with passing years. How much bone density is left at the time of evaluation is a reflection of the net balance left after formation and resorption. In essence, it is your bone bank balance. BMD is evaluated using either x-ray absorption or ultrasound techniques. The most common device used currently is the DEXA®.  DXA®, or DEXA®, involve dual energy x-ray absorptiometry of the hip, femoral neck and lumbar spine. DEXA® is the most common of the BMD® techniques. This utilizes low level x-rays. pDXA® or peripheral DEXA® involves dual energy absorptiometry of peripheral sites such as the finger, forearm and heel. SXA® indicates single x-ray absorptiometry involving the heel. QUS® is quantitative ultrasound of the heel. QCT® involves the use of quantitative CT of the lumbar spine while pQCT® is a peripheral multiple slice technique involving the wrist.

All of these techniques report BMD for the specific sites they measure as a T score and a Z score. The T score describes the patient’s bone mass relative to the average peak bone mass for normal young adult women. No T scores have been established as of yet for men. The patient’s findings relative to the normal findings are expressed as the number of standard deviations (SD). For each SD loss in bone density, the risk of fracture doubles. A 1-to-2.5 SD below normal is considered low bone mass or osteopenia, while a > 2.5 SD below normal is defined as osteoporosis. The Z score compares the patient to others of the same age. Since we want comparisons with normal bone, we use the T score, not the Z score.

 

Excessive Resorption

Excessive bone resorption can occur as part of aging, or it can be secondary to medical diseases e.g. diabetes, alcoholism, hyperthyroidism, hyperparathyroidism, breast and prostate cancers, or the use of medications such as steroids and dilantin. We suggest your bone integrity status be evaluated with a baseline bone mineral density (BMD) to determine your bone mass and also with a first or second morning urine to measure the collagen breakdown product deoxypyridinolium (Dpd) which is commercially available as Pyrilinks-D® (Figure 4).

Figure 4: PYRILINKS-D: Deoxypyridinolium Crosslinks in Urine

If either or both of these are abnormal, it would be good medicine to correct this by stopping excessive bone resorption and aiding bone formation. How do you do this?

 

How To Prevent Excessive Bone Resorption

Calcium supplements help make healthy bone and stop resorption. BPs drive calcium into the bone. If no calcium supplement is given or if calcium is not present during these times, hypocalcemia occurs and poor quality bone is formed. Therefore, when using BPs, start calcium supplements a day or two before initiating BP therapy. Use calcium citrate for better absorption. Calcium by itself has been shown to reduce bone resorption. This is especially true if calcium is administered in the evening, ideally before sleep. Due to the large size of the calcium supplements, we suggest you take 500 mg with dinner and 500 mg at bedtime. Blumsohn et al have described the circadian rhythm of calcium absorption as shown below.

  • Nocturnal increase in parathormone (PTH)
  • Peak Excretion of Dpd & Ntx at 0300–0700
  • Calcium taken in evening suppresses nocturnal increase in PTH
  • Calcium supplements taken in evening suppresses daily excretion of Dpd by 20%, Ntx by 18%.

Citrical® by Mission Pharmaceuticals or Calcium Citrate® by Solgar are two excellent brands of calcium citrate. If your diet is high in calcium, decrease the calcium supplements accordingly and work with your doctor to optimize calcium administration.

Synthetic Vitamin D

Not to be confused with ordinary Vitamin D3, synthetic Vitamin D (1,25 DihydroxyCholecalciferol), has many interesting properties for PC. In a recent issue of The Prostate Forum, an excellent newsletter published by Snuffy Myers MD and his staff, Vitamin D was reviewed. In the area of bone integrity, synthetic Vitamin D (Rocaltrol® or Calcitriol®), can be used to enhance calcium absorption from the gastrointestinal tract. Rocaltrol® also has antiproliferative36-39 and anti-angiogenesis effects40-41 on prostate cancer growth and is able to slow the rate of PSA rise in patients with early recurrent PC.42 The limiting factor in this study was the finding of increased urinary excretion of calcium. We would speculate that the administration of calcium at night, as well as the use of BPs to drive calcium into bone formation, would decrease these findings and allow for higher doses of Rocaltrol®. Rocaltrol® should also be given at bedtime to decrease urinary calcium excretion.

It appears that calcium and Vitamin D have a circadian rhythm which obviously affects their biologic function. Perhaps bone formation and resorption occur mostly at night or in the early hours of the morning. This may be the reason for the complaint of growing pains of teenagers occurring at night, and the need to check the Pyrilinks-D® test with a first or second morning voided urine specimen.

 

Administering Bisphosphonates

For patients with prostate cancer with evidence of bone metastases detected by bone scan or by bone marrow examination using a monoclonal antibody to detect micro-metastatic disease (Impath), we suggest the use of pamidronate (Aredia®). This is given intravenously. We like to give the first dose at 30 mg over 1.5 hours to minimize the chance of an acute phase response (APR). The APR is usually associated with fever within 28-36 hours of the initial exposure to the ABP. The APR is felt to be due to a first-time reaction of the amino BPs with macrophage-like cells resulting in the release of interleukin-1 (IL-1).33 Since we have seen two patients in consultation with kidney damage after a high first dose of Aredia®, we routinely give the first dose at 30 mg and then increase to 60-90 mg every two weeks thereafter.

Serum calcium levels should be watched and the patient encouraged to take calcium supplements as discussed previously. In patients without bone metastases, the use of alendronate (Fosamax®) is encouraged. Since Fosamax® is poorly absorbed in the small intestine it should be given one hour before breakfast and taken only with water. The patient is advised not to lie down after taking Fosamax®. If symptoms of gastrointestinal upset such as belching and burping or discomfort in the stomach region occur, Fosamax® should be stopped and the physician notified.

A key paper on the use of bisphosphonates to reduce new metastases to bone, liver and lung as well as to prolong survival in women with breast cancer was recently published in the New England Journal of Medicine.34 Breast cancer and prostate cancer have strong similarities sufficient to warrant extrapolating data from the breast cancer literature and seeing if such approaches are effective in prostate cancer. In this study, the bisphosphonate used was oral Clodronate® at a dose of 400 mg four times per day. The patients studied included 302 women with breast cancer with tumor cells in the bone marrow. Patients were randomized to Clodronate® (157) versus control (145). Patients in both groups received standard surgical, hormonal and chemotherapy treatments. The results are shown in Table 2 below.

Table 2: Breast Cancer Response to Bisphosphonate

During the median observation period of 36 months, distant metastases (bone or visceral) were detected in 21 women in the Clodronate® group as contrasted to 42 women in the control group. In this study, all women had evidence of bone marrow metastases using immuno-histochemical staining of bone marrow aspirates. Details are shown in the table. The results of this paper should prompt a similar study in prostate cancer.

An alternative agent to be used is (Miacalcin®) nasal spray. Miacalcin® is a derivative of salmon calcitonin. Miacalcin® will reduce bone resorption due to prostate cancer,35 but there are not as many papers on the use of Miacalcin® in regard to bone physiology of PC as there are dealing with the bisphosphonates. Randomized studies should be done since Miacalcin® is so much easier to take than Fosamax®. Miacalcin® is dosed as one spray in one nostril per day with the right and left nostrils alternated to prevent nasal irritation. We have seen just one allergic reaction to Miacalcin® occurring in a patient with a history of fish allergy.

 

Bone Integrity – Concluding Remarks

In my opinion, the institution of bone integrity measures as detailed in this issue of Insights should be a routine part of the management of the PC patient. This is true not only to prevent bone metastases, but also to maintain the structural integrity of the bone to avoid fractures and bone pain.

We will continue to watch the literature on bone integrity in PC since it represents an avenue to increased supportive care of the patient and insights into better tumor control.

References

1. Clarke NW, McClure J, George NJR: The effects of orchidectomy on skeletal metabolism in metastatic prostate cancer. Scand J Urol Nephrol 27:475-483, 1993.

2. Eriksson S, Eriksson A, Stege R, et al: Bone mineral density in patients with prostatic cancer treated with orchidectomy and with estrogens. Calcif Tissue Int 57:97-99, 1995.

3. McGrath SA, Diamond T: Osteoporosis as a complication of orchiectomy in 2 elderly men with prostatic cancer. J Urol 154:535-536, 1995.

4. Daniell HW: Osteoporosis after orchiectomy for prostate cancer. J Urol 157:439-444, 1997.

5. Stepan JJ, Lachman M, Zverina J et al: Castrated men exhibit bone loss: effect of calcitonin treatment on biochemical indices of bone remodeling. J Clin Endocrinol Metab 69:523-527, 1989.

6. Goldray D, Weisman Y, Jaccard N, et al: Decreased bone density in elderly men treated with gonadotrophin-releasing hormone agonist decapeptyl (D-Tryp6-GnrH). J Clin Endocrinol Metab 76:288-290, 1993.

7. Diamond T, Campbell J, Bryant C, et al: The effect of combined androgen blockade on bone turnover and bone mineral densities in men treated for prostate cancer. Cancer 83:1561-6, 1998.

8. Townsend MF, Sanders WH, Northway RO, et al: Bone fractures associated with luteinizing hormone-releasing hormone agonists used in the treatment of prostate cancer. Cancer 79:545-50, 1997.

9. Clarke NW, McClure J, George NJR: Preferential preservation of bone mineralisation by LHRH agonists in the treatment of metastatic prostate cancer. Eur Urol 19:114-117, 1991.

10. Kasperk CH, Wergendal JE, Farley JR, et al: Androgens directly stimulate proliferation of bone cells in vitro. Endocrinology 124:1576-1578, 1989.

11. Manishen WJ, Sivananthan K, Orr FW: Resorbing bone stimulates tumor cell growth: a role for the host microenvironment in bone metastases. Am J Pathol 123:39-45, 1986.

12. Dodwell DJ: Malignant bone resorption: cellular and biochemical mechanisms. Ann Oncol 3:257-67, 1992.

13. Mundy GR: Cytokines and local factors which affect osteoclast function. Int J Cell Cloning 10:215-22, 1992.

14. Thomson BM, Saklatvala J, Chambers TJ: Osteoblasts mediate interleukin-I responsiveness of bone resorption by rat osteoclasts. J Exp Med 164:104-12, 1986.

15. Southby J, Kissin MW, Danks JA, et al: Immunohistochemical localization of parathyroid hormone-related protein in human breast cancer. Cancer Res 50:7710-16, 1990.

16. Averbuch SD: New bisphosphonates in the treatment of bone metastases. Cancer 72:3443-52, 1993.

17. Diel IJ, Solomayer E, Costa SD, et al: Reduction in new metastases in breast cancer with adjuvant clodronate treatment. N Eng J Med 339:357-63, 1998.

18. Mundy GR: Mechanisms of osteolytic bone destruction. Bone 12:Suppl 1:S1-S6, 1991.

19. Mundy GR, Martin TJ: Pathophysiology of skeletal complications of cancer. In: Mundy GR, Martin TJ, eds. Physiology and pharmacology of bone. Berlin, Germany: Springer Verlag, 641-71, 1993.

20. Diel IJ, Solomayer E, Costa SD, et al: Reduction in new metastases in breast cancer with adjuvant clodronate treatment. N Eng J Med 339:357-63, 1998.

21. Coukell AJ, Markham A: Pamidronate. A review of its use in the management of osteolytic bone metastases, tumour-induced hypercalcemia and Pagetís disease of bone. Drugs & Aging 12(2):149-168, 1998. (Figure 2 modified for Insights)

22. Savarese DMF, Valinski H, Quesenberry P, Savarese T: Expression and function of colony-stimulating factors and their receptors in human prostate carcinoma cell lines. Prostate 34:80-91,1998.

23. Nelson JB, Hedican SP, George DJ, et al: Identification of endothelin- 1 in the pathophysiology of metastatic adenocarcinoma of the prostate. Nature Med 1:944-949, 1995.

24. Boonekamp PM, van der Wee-Pals LJA, van Wijk-van Lennep MML, et al. Two modes of action of bisphosphonates on osteoclastic resorption of mineralized matrix. Bone Miner 1:27-39, 1986.

25. Evans CE, Braidman IP. Effects of two novel bisphosphonates on bone cells in vitro. Bone Miner 26: 95-107, 1994.

26. Hughes, DE, Wright KR, Uy HL, et al: Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. J Bone Miner Res 10:1478-87, 1995.

27. Sahni M, Guenther HL, Fleisch H, et al. Bisphosphonates act on rat bone resorption through the mediation of osteoblasts. J Cli Invest 91: 2004-11, 1993.

28. Sahni M, Guenther HL, Fleisch H et al: Bisphosphonates act on rat bone absorption through the mediation of osteoblasts. J Clin Invest 91:2004-2011, 1993.

29. Coleman R, Vinholes J, Purohit 0, et al: Effects of pamidronate on tumour marker levels in breast and prostate cancer -correlation with clinical and biochemical response [abstract no. 1179]. Proc Am Soc Clin Oncol 16: 330a, 1997.

30. Shipman CM, Rogers MJ, Apperley JF, et al: Bisphosphonates induce apoptosis in human myeloma cell lines: a novel anti-tumor activity. Brit J Haem 98:665-672, 1997.

31. van der Pluijm G, Vloedgraven H, van Beek E, et al: Bisphosphonates inhibit he adhesion of breast cancer cells to bone matrices in vitro. J Clin Invest 1; 98:698-705, 1996.

32. Blumsohn A, et al:The effect of calcium supplementation on the circadian rhythm of bone resorption. J Clin Endocrinol Metab 79:730-5, 1994.

33. Adami S, Bhalla AK, Dorizzi R et al: The acute-phase response after bisphosphonate administration. Calcif Tissue Int 41:326-331, 1987

34. Diel KJ, Solomayer E, Costa SD, et al: Reduction in new metastases in breast cancer with adjuvant clodronate treatment. N Engl J Med 339:357-63, 1998.

35. Percival RC, Urwin GH, Harris S, et al: Biochemical and histological evidence that carcinoma of the prostate is associated with increased bone resorption. Eur J Surg Oncol 13:41-49, 1987.

36. Zhuang S, Burnstein KL: Antiproliferative effect of 1a,25- dihydroxyvitamin D3 in human prostate cancer cell line LNCaP involves reduction of cyclin-dependent kinase 2 activity and persistent G1 accumulation. Endocrinology 139:1197-1207, 1998.

37. Getzenberg RH, Light BW, Lapco PE, et al: Vitamin D inhibition of prostate adenocarcinoma growth and metastasis in the Dunning rat prostate model system. Urology 50:999-1006, 1997.

38. Peehl DM, Skowronski RJ, Leung GK, et al: Anti-proliferative effects of 1,25 dihydroxyvitamin D3 on primary cultures of human prostate cells. Cancer Res 54:805-810, 1994.

39. Schwartz GG, Hill CC, Oeler TA, et al: 1,25 dihydroxy-16ene-23-yne-vitamin D3 and prostate cancer cell proliferation in vivo. Urology 46:365-369,1995.

40. Oikawa T, Hirotani K, Ogasawara H, et al: Inhibition of angiogenesis by vitamin D3 analogues. Eur J Pharm 178:247-250, 1990.

41. Oikawa T, Yoshida Y, Shimamura M, et al: Antitumor effect of 22- oxa-1a,25-dihydroxyvitamin D3, a potent angiogenesis inhibitor, on rat mammary tumors induced by 7,12-dimethylbenz[a]anthracene. Anti-cancer Drugs 2:475-480, 1991.

42. Gross C, Stamey T, Hancock S, Feldman D: Treatment of early recurrent prostate cancer with 1,25-dihydroxyvitamin D3 (Calcitriol). J Urol 159:2035-2040, 1998.