CATHEPSIN-D AND TNF-a IN BLADDER CANCER

In a study of 34 normal healthy controls, 35 patients with urinary tract bilharziasis and 93 bladder cancer patients (62 of them are operable cases and 31 are non-operable ones) , serum tumor necrosis factor alpha (TNF-a) and cytosolic Cathepsin-D were estimated. Though both potential markers were elevated in bladder cancer patients, neither Cathepsin-D nor TNF-a showed associations of prognostic value since there were no positive correlations with tumor stages, grades or association of tumors with bilharzia) ova or lymph node involvement.

In 37, bilharzial ova were found in their tissues while 25 were free.
Group B. Urinary tract bilharzial patients (N=35) were aged from 25-65 years (mean=45).This group included 25 males and 10 females.Ten were heavy smokers, 15 light smokers and J 0 nonsmokers.These patients were diagnosed by clinical examination and serological test for bilharziases.Cancer of urinary bladder was excluded by urine cytology and cytoscopy.
Group C. Normal healthy controls (N=34) were aged from IS-65 years (mean=2S).This group included 30 males and four females.Nine were heavy smokers, 10 were light smokers and 15 were non-smokers.They were found healthy by clinical examination and all were checked with serological test for bilharziases giving results within the normal range.
Blood Samples. 10 m!. venous blood was withdrawn from all individuals.Serum was obtained by centrifugation at 4000 rpm.divided into aliquots and stored at -SO°C until assayed.For the bladder cancer group, blood samples were taken 1-3 days preoperati vel y.
Tissues Samples.Specimens of bladder tumor tissues were obtained directly at operation.Blood was washed away by ice cold saline.Fat and necrotic tissues were rapidly dissected from the tumor tissue.About 1-2 gm. of tumor tissue was wrapped in aluminium foil and directly frozen at -sonc, while a portion of normal bladder tissue at least 10 cm distant from the tumor was obtained from the same patient to be used as control.for the cancer tissue and treated in a similar manner.For isolation of the cytosol and membrane fraction, certain weights of both tumor and control tissues were chopped with a scalpel and scissors.The minced tissues were homogenized, by Teflon glass homogenizer, in 10 volumes of ice cold phosphate buffer.Tissue homogenate was centrifuged at SOO xg for 10 minutes to precipitate the nucleus.The supernatant was separated and ultracentrifuged at 100.000 xg for 30 minutes.The cytosol supernatant was divided into aliquots and the membrane pellet was resuspended in one m!. of the homogenization buffer with the aid of sonicator and ultracentrifuged at 100.000 xg for 30 minutes.The membrane fraction in the supernatant was divided into aliquots.Both cytosol and membrane-enriched fraction aliquots were frozen at -SOnC until assayed.
Serological assay for bilharziasis was done using the indirect haemaggiutination reagent supplied by Behringwerke AG, Germany.Serum TNF-C( was determined according to the principle of Tracey et al. (l9S7) using the reagent supplied by Medgenix Diagnostics, Belgium.It is a solid phase immunoassay on microtitre plate.Cathepsin-D was estimated in the cytosol fraction according to the principle of Predine (1992) using the reagents supplied by CIS Bio International, France.The Cathepsin-D concentration was expressed per mg tissue protein.Protein concentration was estimated according to the principle of Bradford (1976).It depends on the reaction of protein and coomassie brilliant blue dye which leads to change to colour from blue to green measured colorimetrically at 595 nm.

RESULTS
The findings are presented in Tables 1-6.

DISCUSSION
Schistosomiasis is one of the major public health problems in Egypt.It is one of the predisposing factors for bladder cancer which is the most common malignant tumor among Egyptians (El-Bolkainy et aI., 1981).
Tumor markers can be of diagnostic and prognostic value.The ultimate serum concentration of tumor markers reflects a multifactorial dynamic equilibrium between marker synthesis and clearance (pohl, 1990).Cathepsin-D is an acidic lysosomal aspartyl endoprotease with at least two biologic properties: estrogen and/or growth factordependent mitogenic activity and enzymatic activity on proteoglycans, extracellular matrix and basement membrane (Vignon etal., 1986, Briozzo et al., 1988and Rochefort, 1990).These properties suggest a potential role for Cathepsin-D in the invasion and metastasis of cancer cells (Liotta, 1988).Previous studies reported significant correlations between Cathepsin-D concentration and prognosis in breast cancer.High concentration of total Cathepsin-D was regarded to be a marker for poor prognosis (Thorpe et aI., 1989 andBrouillet et al., 1993).
The present work showed a significant elevation in malignant cytosolic Cathepsin-D when compared with that of normal tissue.This agrees with Reid et al. (1986), who reported that Cathepsin-D is expressed in bladder carcinoma.This may be attributed to the loss of regulation and overexpression of proteins involved in the control of cell proliferation and migration.Alternatively, a protease acting normally in lysomes may be secreted excessively from the cancer cells because of an altered processing of it precursor (Capony et ai., 1989)  Despite its elevated level in cancer tissue, in the present study, at 100% and 90% specificity, cytosol Cathepsin-D has a sensitivity of only 35.7% and 59.5 % respectively.Cathepsin-D level is independent of clinical stage, pathological grade, lymph node involvement and bilharziasis.This is in agreement with Namer et ai. (1989), Marsigliante et al. (1994) , Scorilar et al. (1994) and Seshadri et al. (1994) but contrary to Foekins et al. (1993) and Hahnel et al. (1994) who found significant correlations between Cathepsin-D level and lymph node involvement with tumor size.We observed that cytosolic Cathepsin-D is more strikingly elevated in transitional cell carcinoma than squamos cell carcinoma; the reason for this is unclear.
Tumor necrosis factors play an important role in the defence against viruses, tumors and bacteria.Evidence is also accumulating that they may playa role in the pathogenesis of several diseases, including autoallergies such as experimental allergic encephalomyelitis and viral infections such as AIDS (Ruddle, 1986).Concerning TNF-a, the present work revealed significant elevations of serum TNF-a in patients with bladder cancer and those with urinary tract bilharziasis.No significant difference was observed either between cancer and bilharzial groups or between operable and non-operable cases of bladder cancer.As a tumor marker, serum TNF-a showed the highest sensitivity for nonoperable cases.This may be attributed to the highly advanced cancer state of those patients.The high "false positivity" obtained with urinary tract bilharzial patients may be related to the excessive release of TNF-a by cells of the immune system in response to various invasive stimuli.These results agree with Balkwill et (I/. (1987) and Owen-Schaub et al. (1988).Our findings also suggest that serum TNF-a is independent of cancer type, pathological stage and grade, involvement or not of lymph nodes and association of the tumor with bilharzial ova or not.
We conclude that TNF-a can not be used for the differential diagnosis between bilharzial and bladder cancer patients.Overexpression of Cathepsin-D was found in tumor tissue compared to normal tissue of the bladder.Nevertheless , neither Cathepsino orTNF-a were of prognostic value since there were no positive correlations with tumor stage, grade, of association of tumors with bilharzial ova or lymph node involvement.

Table 2 .
Sensitivity of cytosol Cathepsin-D in bladder cancer patients at different specificity.

Table 3 .
Mean value and sensitivity of cytosol Cathepsin-D according to cancer type, pathological stage, grade, involvement of lymph nodes and bilharziasis.

Table 4 .
Serum TNF-a (pg/ml.) in the different studied groups.

Table 5 .
Sensitivity of serum TNF-a in bladder cancer patients and false positivity urinary tract bilharzial patients.

Table 6 .
Mean value and sensitivity of serum TNF-a according to cancer type, stage, grade, involvement of lymph nodes and bilharziasis.