Cachexia often causes deterioration in the quality of life in cancer patients; however, its mechanism remains poorly understood. Cachexia has often been observed in experimental animals with bone metastases, and parathyroid hormonerelated protein (PTHrP) plays an important role in the formation of such metastases. We therefore investigated the possible involvement of PTHrP in an experimental cachexia model using human lung-cancer cells (HARA-B Cachexia often accompanies various cancers in advanced stages and tends to deteriorate the quality of life in such patients while also affecting the survival period and/or response to chemotherapy. 1 As a result, understanding the mechanism of cancer cachexia could lead to the development of appropriate types of therapeutic intervention for these patients; however, its mechanism remains poorly understood. Cachexia is thought to be induced by soluble factors produced and released from hematopoietic cells and/or cancer cells. Several cytokines, i.e., TNF, 2,3 IL-1, 4 IFN-␥, 5 IL-6, 6,7 leukemia inhibitory factor (LIF) 8 and TGF-␤, 9 induce cachexia in different models. However, exactly how these cytokines are involved in the mechanism of cachexia induction remains to be elucidated.In the present report, we describe an experimental cachexia model using a human lung cancer-derived cell line (HARA-B), in which parathyroid hormone-related protein (PTHrP) mRNA is strongly expressed. Using this model, the involvement of PTHrP in the mechanism of cachexia induction was investigated. MATERIAL AND METHODS CellsHARA-B cells were established from a bone lesion formed after the intracardiac inoculation of HARA cells. 10 Cells were maintained in RPMI-1640 supplemented with 10% FBS at 37°C in humidified atmosphere of 5% CO 2 . Cells were grown as a monolayer culture in a flask, and a single-cell suspension of cells was obtained by trypsin treatment. MiceMale 5-week-old BALB/c nu/nu mice (Nippon CLEA, Tokyo, Japan), kept in a specific pathogen-free environment, were used. Measurement of cachexic parametersFor the in vivo experiment on cachexia, a single-cell suspension of HARA-B cells (5 ϫ 10 6 ) was inoculated s.c. into the right flank of nude mice. Body weight and tumor size [length (a) and width (b)] were thereafter measured periodically. Then, mice were killed 8 weeks after cell inoculation. At this time, blood was collected from the heart and the epididymal adipose tissue and gastrocnemius muscle were weighed. Serum samples, obtained after centrifugation, were stored at -30°C until serum glucose, calcium and PTHrP levels were determined. Serum glucose and calcium levels were measured using the enzyme-reaction method (glucose C⌸ test; Wako, Osaka, Japan) and a color-chelate reaction method (Calcium C-test, Wako), respectively. The serum PTHrP level was measured using a radioimmunoassay kit specific for the C-terminal portion of PTHrP (Daiichi, Tokyo, Japan). The serum mouse IL-6 level was measured using an ELISA kit (Endogen, Woburn, MA). Tumor weight was estimated by the formula (ab 2...

Nude rats bearing the LC-6-JCK human lung cancer xenograft displayed cancer-associated wasting syndrome in addition to humoral hypercalcemia of malignancy. In these rats, not only PTHrP but also several other human proinflammatory cytokines, such as IL-6, leukemia-inducing factor, IL-8, IL-5 and IL-11, were secreted to the bloodstream. Proinflammatory cytokines induce acute-phase reactions, as evidenced by a decrease of serum albumin and an increase in a1-acid glycoprotein. Tumor resection abolished the production of proinflammatory cytokines and improved acute-phase reactions, whereas anti-PTHrP antibody affected neither proinflammatory cytokine production nor acutephase reactions. Nevertheless, tumor resection and administration of anti-PTHrP antibody similarly and markedly attenuated not only hypercalcemia but also loss of fat, muscle and body weight. Body weight gain by anti-PTHrP antibody was associated with increased food consumption; increased body weight from antiPTHrP antibody was observed when animals were freely fed but not when they were given the same feeding as those that received only vehicle. Furthermore, nude rats bearing LC-6-JCK showed reduced locomotor activity, less eating and drinking and low blood phosphorus; and anti-PTHrP antibody restored them. Although alendronate, a bisphosphonate drug, decreased blood calcium, it affected neither locomotor activity nor serum phosphorus level. These results indicate that PTHrP represses physical activity and energy metabolism independently of hypercalcemia and proinflammatory cytokine actions and that deregulation of such physiologic activities and functions by PTHrP is at least in part involved in PTHrP-induced wasting syndrome. ' 2005 Wiley-Liss, Inc.Key words: parathyroid hormone-related protein; wasting; proinflammatory cytokine; acute-phase reaction; locomotor activity Cancer-associated wasting (cancer wasting) is a complex disease often associated with anorexia and cachexia and characterized by several metabolic and behavioral abnormalities, such as early satiety, weakness, fatigue, depression and weight loss. Cancer wasting occurs in about 10% of advanced cases of malignancy. It correlates with poor prognosis irrespective of tumor mass or tumor metastases 1 and affects the susceptibility to and tolerability of chemotherapy.2 Furthermore, cancer wasting is not easily resolved by forced caloric intake.3 Several factors are thought to be involved in cancer wasting. TNF-a, IL-1b, IL-6, LIF and IFN-c have been demonstrated to cause cachectic symptoms in animal models. 4 Activin, a member of the TGF-b gene family, was found to induce cachectic symptoms in mice.5 PIF, a sulfated glycoprotein that induces protein catabolism in isolated muscle cells, has been detected in serum samples from cachectic mice and from cancer patients.1 LMF, a homologue of the plasma Zn-a2 glycoprotein which induces lipid mobilization and catabolism in rats, has been isolated from the urine of cancer patients. 6 Nevertheless, the etiology of cancer cachexia is still not well...

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Purpose: Parathyroid hormone-related protein (PTHrP) is a causative factor of humoral hypercalcemia of malignancy (HHM) and concurrent anorexia and wasting. Because changes in the expression of hypothalamic feeding-regulating peptides can directly affect appetites and thereby can cause anorexia and wasting, we addressed whether the cachectic syndromes induced by PTHrP rely on the action of hypothalamic feeding-regulating peptides. Experimental Design: Rats were inoculated with a LC-6 human cancer xenograft that secreted PTHrP, and the mRNA levels of the hypothalamic feeding-regulating peptide genes and serum leptin levels were examined before and after the development of HHM by in situ hybridization histochemistry and ELISA, respectively. Some rats were given the anti-PTHrP antibody. Results and Conclusion: The mRNA levels for the orexigenic peptides, such as the agoutirelated protein and the neuropeptide Y in the arcuate nucleus (Arc), were significantly increased after the development of HHM, whereas the mRNA levels for the anorexigenic peptides, such as the proopiomelanocortin in the Arc, the cocaine and amphetamine-regulated transcript in the Arc, and the corticotropin-releasing factor in the paraventricular nucleus, were significantly decreased after the development of HHM. Plasma leptin levels were also reduced in cachectic rats, and the administration of anti-PTHrP antibody to the cachectic rats not only improved the cachectic symptoms but also restored the mRNA levels of these orexigenic and anorexigenic peptides, except for orexin. Thus, PTHrP induces HHM and concurrent cachectic syndromes by mechanisms other than directly modulating the leptin or hypothalamic feeding-regulated peptides.

Purpose: Bisphosphonate and calcitonin lower blood calcium in humoral hypercalcemia of malignancy (HHM) by suppressing osteoclastic bone resorption, but repeated administration of these drugs often leads to relapse. In this study, we examined the roles of parathyroid hormoner elated protein (PTHrP) in the development of bisphosphonate-and calcitonin-refractory HHM. Experimental Design: Nude rats bearing the LC-6 JCK tumor xenograft (LC-6 rats) exhibited high bone turnover and HHM. Repeated administration of alendronate induced a sustained suppression of the bone resorption, but it caused only early and transient reduction of the blood calcium levels, leading to unresponsiveness to the drug. Because high blood levels of PTHrP were detected in the LC-6 rats, those that developed alendronate-refractory HHM were treated with an anti-PTHrP antibody. Results: Administration of anti-PTHrP antibody to animals that received repeated administration of alendronate, thereby developing alendronate-refractory HHM, resulted in an increase in fractional excretion of calcium and a marked decrease of blood calcium level. Drug-refractory HHM was also observed in animals that received another osteoclast inhibitor, an eel calcitonin analogue elcatonin. The blood calcium level decreased after the initial administration of elcatonin, but it eventually became elevated during repeated administration. Administration of the anti-PTHrP antibody, but not of alendronate, effectively reduced the blood calcium of the animals that developed elcatonin-refractory HHM. Conclusion: High levels of circulating PTHrP and the resulting augmentation of renal calcium reabsorption is one of the major causes of the emergence of osteoclast inhibitor-refractory HHM. Thus, blockage of PTHrP functions by a neutralizing antibody against PTHrP would benefit patients who develop bisphosphonate-or calcitonin-refractory HHM.Humoral hypercalcemia of malignancy (HHM) is one of the most common paraneoplastic syndromes that considerably deteriorate the quality of life of cancer patients (1). Increased bone resorption has been thought to play important roles in the development of HHM and current therapies for HHM focus rather on inhibiting the osteoclastic bone resorption. In fact, bisphosphonate drugs that interfere with the osteoclasts have become the gold standard for the treatment of HHM (2). However, there are patients who do not respond to these drugs and others often suffer relapse after repeated administration of bisphosphonate drugs. In a recent study by Body et al. (3), the percentage of patients who responded to a first dosing of pamidronate was 90%, but decreased to 80% and 70% at second and third dosings, respectively. Furthermore, the response rate to pamidronate was higher in patients with lower blood parathyroid hormone -related protein (PTHrP) levels (between 2 and 12 pg/mL) than those with higher PTHrP levels (higher than 12 pg/mL), suggesting the involvement of PTHrP in bisphosphonate-refractory HHM (4).Bisphosphonate-refractory HHM has also ...