Chi-Wei Lin

Photodynamic therapy with hematoporphyrin derivative in the treatment of superficial transitional-cell carcinoma of the bladder

Photodynamic therapy involves light-induced destruction of tumors containing a photosensitizer such as hematoporphyrin derivative. We conducted a collaborative study to evaluate the efficacy of this form of therapy in treating superficial transitional-cell carcinoma of the bladder. Thirty-seven patients were evaluated and 20 were selected for treatment. A total of 50 papillary tumors and 3 areas of carcinoma in situ were treated. All except two tumors were smaller than 2.5 cm. Assessments for treatment response and toxicity were carried out three months after treatment. The initial diagnosis of one patient was revised after the biopsy material was reviewed, and this patient was not included in the analysis. Complete eradication of all tumors was observed in 9 of 19 patients (47 percent), including those with carcinoma in situ. In the remaining 10 of these 19 patients, 13 tumors could not be eradicated (the overall eradication rate was 37 of 50 tumors [74 percent]), but 9 of the 10 patients had a reduction in tumor size, number, or both of 50 percent or more. We conclude that photodynamic therapy is useful in the treatment of superficial transitional-cell carcinoma of the bladder, but controlled trials will be required to define its place in the treatment of cancer.

Evaluation of Prostate Specific Acid Phosphatase and Prostate Specific Antigen in Identification of Prostatic Cancer

The peroxidase-anti-peroxidase technique was used to stain for prostate specific acid phosphatase and prostate specific antigen in 12 patients with primary tumors and in 12 patients with metastases in whom the nature of the tumor was in doubt after routine histopathological studies. Nine of the primary tumors were positive for both markers and an additional 2 tumors stained for prostate specific antigen only. Six metastatic lesions stained for both markers and a seventh for prostate specific antigen alone. Thus, 11 of 12 primary tumors and 7 of 12 metastases studied were proved to be of prostatic orgin. While the peroxidase staining was sometimes weak and uneven this method, using prostate specific antigen and prostate specific acid phosphatase, allowed for ready identification of metastases. The heterogeneity of the tumors in regard to these 2 prostate markers is demonstrated, and the value of staining for prostate specific acid phosphatase and prostate specific antigen is emphasized.

ANDROGEN RESPONSIVENESS OF STROMAL CELLS OF THE HUMAN PROSTATE: REGULATION OF CELL PROLIFERATION AND KERATINOCYTE GROWTH FACTOR BY ANDROGEN

PURPOSE Growth and development of the prostate are androgen dependent and mainly influenced by stromal-epithelial interaction. It is believed that indirect androgenic activation of paracrine factors like keratinocyte growth factor (KGF) in the prostatic stroma influences the growth of epithelial cells. In this study we investigated the role androgen plays in stromal cell growth and stimulation of KGF in the human prostate. MATERIALS AND METHODS Stromal cells were derived from explant primary culture of human normal or benign prostatic tissue. The effect of different dihydrotestosterone (DHT) concentrations on cell proliferation was measured using 3[H]thymidine incorporation assay. The effect of DHT on levels of KGF protein was determined by Western blotting. The effect of DHT on levels of KGF gene expression was measured by various cycles of polymerase-chain-reaction (PCR) and multiplex PCR. RESULTS Characterization of stromal cells showed epithelial cells less than 9.5% in all passages. DHT stimulated human prostate stromal cells in a dose dependent fashion over a concentration range of 0.001-10 nM. Immunocytochemical evaluation of KGF after DHT exposure showed a higher staining intensity. Relative quantitation of Western blotting showed a 1.93-fold increase in KGF protein in the androgen treated stromal cells. At 1 nM DHT conventional and multiplex PCR revealed a significant stimulation of the KGF mRNA expression. CONCLUSIONS These data show for the first time that androgen stimulates cell proliferation as well as KGF protein and gene expression in human prostate stromal cells. This supports the hypothesis that androgen-induced stromal-derived KGF stimulates prostate epithelial cell growth.

Intratumor Injection as a More Effective Means of Porphyrin Administration for Photodynamic Therapy

Photodynamic therapy (PDT) with hematoporphyrin derivative (HpD) as the photosensitizer is a promising new cancer treatment. The major drawback of this procedure is the resulting skin photosensitivity. Patients must remain in subdued light for four to six weeks to avoid cutaneous phototoxicity. In this study, we examine the possibility of reducing the skin photosensitization while maintaining the tumor phototoxic effect by administering the drug directly into the tumor. A subcutaneously implanted mouse bladder tumor (MBT-2) was used. HpD was administered either intraperitoneally (I.P.; 20 mg./kg. b.w.) or by intratumor injection (I.T.; 0.4 mg./cc tumor). The concentrations of HpD in tumor and various tissues (skin, muscle, liver, spleen, kidney, bladder and whole blood) were analyzed at various times after the injection, by 3H-HpD method and by a fluorometric method. Results indicated that at three to 96 hours after the administration, porphyrin levels in tumor were about three to 15 times higher by I.T. than by I.P. injection, while the concentrations in skin and other tissues were 1.3 to 10 times lower. Consequently, at 24 hours after injection ratios between tumor to skin porphyrin were 14 to 92 times higher for I.T. than I.P. injection. Higher porphyrin levels in tumor and lower in normal tissues would indicate lower skin photosensitivity, systemic cytotoxicity and possible greater tumor photosensitivity. This method of porphyrin administration may be useful for the PDT of certain single lesions that are accessible for direct injection.

Immunolocalization of the keratinocyte growth factor in benign and neoplastic human prostate and its relation to androgen receptor.

Growth and development of the prostate are androgen‐dependent. Keratinocyte growth factor (KGF), widely expressed by mesenchymal cells, is thought to act like an andromedin between stroma and epithelium of the prostate. Since KGF has recently emerged as an autocrine mediator in prostate cancer, we investigated the role KGF plays in the human prostate and its relationship to androgen receptor (AR).

PHOTODYNAMIC DESTRUCTION OF LYSOSOMES MEDIATED BY NILE BLUE PHOTOSENSITIZERS

Previous studies have established that a number of Nile blue derivatives are potent photosensitizers and that they are localized primarily in the lysosomes. The present study examines whether the lysosome is a main target of the photocytotoxic action mediated by these sensitizers. Chosen for this study were NBS‐61 and sat‐NBS, which represented, respectively, derivatives with high and moderate degrees of lysosomal selectivity. Overall results indicated that both derivatives are very effective in mediating a photodestruction of lysosomes. This is indicated by the light‐and drug‐dose‐dependent losses of acid phosphatase staining particles, reduction of hexosaminidase in the lysosomecontaining subcellular fraction, and impairment of the lysosomes to take up and sequester acndine orange. Ultrastructurally, swollen and ruptured lysosomes were seen as one of the first evidences of cell damage mediated by these photosensitizers. However, the study also showed that sat‐NBS, which is less lysosomal selective, was less effective in mediating lysosomal destruction. Also, the degree of lysosomal destruction mediated by sat‐NBS did not parallel the degree of cytotoxicity generated. This implies that for derivatives that are not exclusively localized in the lysosome, other subcellular sites may also be damaged by the photodynamic action and may play a role in the photocytotoxic process.

Vascular invasion as a prognosticator of metastatic disease in nonseminomatous germ cell tumors of the testis. Importance in “surveillance only” protocols

Forty‐five nonseminomatous germ cell carcinomas of the testis were evaluated retrospectively to define the biologic features associated with the occurrence of metastatic disease. A statistical analysis of several pertinent clinical and pathologic factors was performed. The factors evaluated included: duration of symptoms before diagnosis, serum level of alpha‐fetoprotein, serum or urinary level of human chorionic gonadotropin, testicular weight, extent of local tumor (pathologic T stage), and vascular invasion at the primary site. In each case, metastases were documented by a retroperitoneal node dissection, other biopsies, or by chest films. In 29 tumors with vascular invasion, 25 patients were seen with metastatic disease. In 16 tumors without vascular invasion, 3 patients demonstrated metastasis. The presence or absence of vascular invasion was strongly correlated with concomitant lymph node involvement or subsequent appearance of other metastatic disease (chi‐square = 17.19). Additionally, vascular invasion in bifactoral analysis with tumor size and pathologic T stage proved a significant prognosticator even in low‐staged (chi‐square = 8.48) and small tumors (chi‐square = 8.13). The implications of these findings, both as an adjunct to the staging of nonseminomatous germ cell tumors and in the management of clinical Stage I lesions, are discussed.

IDENTIFICATION OF AN ACTIVIN-FOLLISTATIN GROWTH MODULATORY SYSTEM IN THE HUMAN PROSTATE: SECRETION AND BIOLOGICAL ACTIVITY IN PRIMARY CULTURES OF PROSTATIC EPITHELIAL CELLS

PURPOSE To determine if the activin/follistatin system is present in human prostate tissue and primary cultures of prostatic epithelium and if these growth factors play a role in the control of epithelial cell growth. MATERIALS AND METHODS Cells derived directly from human prostates were studied to determine: a) if they secrete activin and follistatin, and b) if they are growth inhibited by activin. Primary cell cultures were established from tissues removed from 13 unselected prostate carcinoma patients in order to examine the secretion of activin and follistatin and test the effects of these proteins on cell proliferation. RESULTS Both primary explant cells and epithelial cells isolated and sub-cultured from explant cultures secreted activin A and follistatin. Treatment of cultured cells with recombinant human activin A resulted in a dose-dependent inhibition of thymidine incorporation, with an IC50 of 0.22 nM. Recombinant follistatin neutralized the inhibitory effects of activin A on cell proliferation whilst adding follistatin alone enhanced thymidine incorporation, suggesting a similar neutralizing effect on the endogenous activin produced by these cells. CONCLUSION These results demonstrate that cells derived from human prostate tissue secrete activin and follistatin and, as observed in human prostate cancer cell lines, activin inhibited the growth of prostatic epithelial cells. Also consistent with our earlier studies of prostate cancer cell lines, the biological activity of activin was neutralized by follistatin. These observations support the hypothesis that the activin/follistatin system plays an important role in the local regulation of human prostate cell growth.

The Effect of Verapamil on a Multi-Drug ResistantBladder Carcinoma Cell Line and Its Potential as anIntravesical Chemotherapeutic Agent

A human bladder transitional cell carcinoma cell line, MGH-U1R, exhibits reproducible resistance to doxorubicin. We examined the effects on survival of this cell line caused by verapamil, which has been shown to reverse multi-drug resistance in vitro in other neoplastic cell lines. Both MGH-U1R and MGH-U1, the non-resistant parent cell line, were treated with varying concentrations of doxorubicin alone, verapamil alone, or both drugs simultaneously, all for one hour. Cells were then grown in drug-free medium for 10 days, stained, and counted. Standard survival curves were calculated. Verapamil alone had no significant cytotoxicity. Verapamil at concentrations of 16 micrograms./ml. and 32 micrograms./ml. decreased the IC50 of doxorubicin for MGH-U1R by a factor of 2.5. Using H3-verapamil, we also examined the systemic and local absorption of this drug resulting from intravesical verapamil administration in rabbits. All animals were treated for one hour, and multiple serum samples were drawn during treatment. Verapamil was found in high concentrations in the mucosa, less in the adventitia, and was absent in venous blood. Verapamil effectively reverses resistance to doxorubicin of MGH-U1R in vitro. The intravesical use of verapamil appears to be safe, and may prove to be a useful adjunct in the intravesical therapy of some bladder tumors.

Establishment and Characterization of a Doxorubicin-Resistant Human Bladder Cancer Cell Line (MGH-U1R)

A doxorubicin-resistant bladder cancer cell line has been established. This was accomplished by exposing an established human bladder tumor cell line, MGH-U1, to progressively higher concentrations of doxorubicin over a period of 12 months. The resistant cells, MGH-U1R, are nine times more resistant to doxorubicin and 30 times more resistant to daunorubicin than the parent cells. The MGH-U1R and the MGH-U1 cells have identical isozyme phenotypes. Compared to the parent cells, the resistant cells have a slower growth rate, lower confluent density, are more heterogeneous morphologically, and exhibit more chromosomal aberrations and rearrangements. The resistant cells may now be used as an experimental system for the search of means to overcome drug resistance in human bladder cancer.