Synergistic anticancer activity of biologicals from green and black tea on DU 145 human prostate cancer cells.

There is considerable interest in the potential of botanicals in preventing and/or alleviating chronic ailments. Among the most studied botanicals are compounds present in green and black teas. Nontoxic tea polyphenols are potent antioxidants, and they also modulate several signalling pathways and inhibit proteins such as MMP-9 or protein plasminogen activator system, making them very attractive potential therapeutics. One criticism of the prophylactic or therapeutic use of green or black tea polyphenols was presumably the poor bioavailability of these chemicals when ingested. However, studies have shown that epigallocatechin-3-gallate (EGCG) and theaflavin (TF) can be detected in the small and large intestine, liver, and prostate of experimental animals after consumption of tea extracts. In particular, a study was carried out on 20 men scheduled for prostatectomy, who were assigned to consume teas for five days before surgery. Tea polyphenols were detected in the prostate. This fact contradicts the common misconception of poor bioavailability of TF and EGCG and makes feasible the application of green or black tea polyphenols as prophylactic and therapeutic agents. Theaflavins and catechins seem to act on cancer cells largely through different pathways, so utilisation of both could offer synergistic anticancer effects, but so far no work has been done on the cumulative effects of EGCG and TF on prostate cancer. Therefore, in this study we have investigated if EGCG in combination with TF can reduce the rate of prostate cancer growth, and we have observed greater cell death compared to application of either TF or EGCG alone.

A comparative study of the inhibiting effects of mitomycin C and polyphenolic catechins on tumor cell implantation/growth in a rat bladder tumor model.

PURPOSE: Mitomycin C (Novaplus®) is often instilled intravesically in the postoperative period to prevent tumor cell implantation/regrowth after transurethral tumor resection. In an earlier study EGCG prevented tumor cell implantation/growth in an experimental bladder tumor model simulating clinical transurethral bladder resection. We compared the efficacy of EGCG (Polyphenon E®) to that of mitomycin C to prevent tumor cell implantation/growth in this model. MATERIALS AND METHODS: Mitomycin C and EGCG were studied for their in vitro and in vivo effects. The AY-27 rat urothelial tumor cell line was used for in vitro and in vivo studies. In vitro cell viability studies included trypan blue exclusion, MTT proliferation assay and clonal growth assay. Fischer 344 female rats were used for intravesical tumor implantation/growth assay using an electrocautery injury model. Tumor growth in vivo was assessed in controls treated with phosphate buffered saline and in bladders treated with mitomycin C or EGCG by standard histological techniques using hematoxylin and eosin 4 weeks after injury. RESULTS: Mitomycin C and EGCG showed cytotoxicity on all in vitro assays. They were equivalent for preventing intravesical tumor growth. CONCLUSIONS: EGCG prevents intravesical tumor growth with efficacy equivalent to that of mitomycin C in this experimental model.

Prostatic involution after intraprostatic injection of cobra toxin.

PURPOSE: We evaluated the comparative effects of intraprostatic injection of cobra cardiotoxin D and botulinum toxin type A on prostate structure in the rat model. MATERIALS AND METHODS: A total of 18 Sprague-Dawley® rats weighing 500 to 600 gm received a single 0.1 ml injection of saline (6), botulinum toxin type A (6) or the cardiotoxin D (6) component of cobra (Naja naja atra) toxin in the right and left ventral lobes of the prostate. At 14 days the rats were sacrificed. The prostate glands were harvested, weighed and processed for immunohistochemical and morphological studies. RESULTS: Prostate glands injected with cardiotoxin D showed significantly decreased weight compared to that of prostates injected with botulinum toxin type A and the saline control. Prostatic atrophy in the glandular component with flattening of the epithelial lining was seen histologically in rats that received botulinum toxin and cardiotoxin D. Each group injected with cardiotoxin D and botulinum toxin showed a significant increase in the number of apoptotic cells compared with controls while only the botulinum toxin group showed a significant increase in the number of proliferating cells. Only rats injected with botulinum toxin had body weight loss. CONCLUSIONS: Our study shows that intraprostatic injection of cobra cardiotoxin D induces prostatic atrophy and leads to a decrease in prostatic weight greater than that of intraprostatic injection of botulinum toxin type A. No systemic effects, such as decreased body weight, were noted after cardiotoxin D injection. Further studies are warranted but the statistically significant decrease in the number of proliferating cells implies a prolonged effect of cardiotoxin D.

Very long half-life plasminogen activator inhibitor type 1 reduces bleeding in a mouse model.

OBJECTIVE: To investigate the potential for the future clinical use of a very long half-life plasminogen activator inhibitor type 1 (VLHL PAI-1) as a haemostatic agent. MATERIALS AND METHODS: We developed a VLHL PAI-1 (half-life >700 h) recombinant mutant of PAI-1 and assessed VLHL PAI-1 for its ability to inhibit fibrinolysis in vitro using human, rabbit, mouse and rat blood. Fibrin clot lysis time, monitored by thromboelastometry, was determined at various concentrations of VLHL PAI-1. Also, we determined total bleeding time and total blood loss of control, VLHL PAI-1-, tissue-type plasminogen activator (tPA)- and tPA + VLHL PAI-1-treated mice. RESULTS: Using a thromboelastometer, mouse blood was most similar to human blood in its coagulation and fibrinolytic characteristics. We evaluated the affect of VLHL PAI-1 on haemostasis using the mouse model and showed that VLHL PAI-1 is an effective inhibitor of fibrin clot degradation. It reduced time of bleeding and total blood loss. CONCLUSION: VLHL PAI-1 may provide an important physiological mechanism to protect clots from premature dissolution in surgical and trauma settings.

Prevention and treatment of transitional cell carcinomatosis with intraperitoneal chemotherapy in a rat model.

PURPOSE: Tumor spillage from bladder perforation during transurethral bladder tumor resection or cystectomy risks seeding the peritoneum with transitional cell carcinoma. We determined the lowest effective mitomycin C dose to prevent tumor implantation and the potential efficacy of delayed therapy. Additionally, we investigated the effect of tumor debulking combined with intraperitoneal mitomycin C. MATERIALS AND METHODS: Using our established murine model of intraperitoneal transitional cell carcinoma implantation mitomycin C was instilled at decreasing concentrations to find the lowest effective dose. To evaluate the effectiveness of delayed therapy mitomycin C was administered on day 3 or 7 after tumor implantation. Finally, surgical debulking of established tumors with or without mitomycin C was performed. RESULTS: All control animals had disseminated carcinomatosis. The lowest effective intraperitoneal mitomycin C dose to prevent implantation was 0.3125 mg/m(2). Administration of mitomycin C on day 3 after instillation resulted in tumor-free status in 50% of the animals, although no rats were tumor-free when treated on day 7. Tumor debulking only for established disease cured 40% of the animals, whereas debulking combined with mitomycin C had a 100% cure rate. CONCLUSIONS: Intraperitoneal mitomycin C prevents tumor growth after transitional cell carcinoma implantation. Delayed therapy is not as effective as immediate treatment but cure is still possible, particularly when combined with surgical debulking, in a rat model.

PAI-1 induces cell detachment, downregulates nucleophosmin (B23) and fortilin (TCTP) in LnCAP prostate cancer cells.

Plasminogen activator inhibitor (PAI-1) is an anticancer agent that inhibits plasmin driven proteolysis, limiting angiogenesis and metastasis. In low concentrations it could induce cancer cell motility by interacting with urokinase (uPA), its receptor (uPAR), vitronectin and integrins. Active PAI-1 binds to uPA forming a complex with uPAR, while the latent form of PAI-1 does not. PAI-1 is found in both forms in the circulation. It is not clear which form acts as an anticancer agent and how it interacts with malignant cells. To investigate how these forms reduce angiogenesis or metastasis, we have created PAI-1 cysteine mutants in the active conformation (VLHL PAI-1) with an extended half-life that reaches approximately 700 h and its R369A mutant, which has an active conformation but cannot bind to uPA (VLHLNS PAI-1). Both VLHL PAI-1s convert into the latent form when treated with a reducing agent (DTT) that breaks disulfide bridges. Unexpectedly, during routine investigation of LnCAP cell proliferation, we have found that cells detach from the culture vessels regardless of PAI-1 conformation or activity. Further investigation showed that treatment of cancer cells with VLHL PAI-1 downregulated nucleophosmin, while all forms of PAI-1 downregulated fortilin. These two proteins are implicated in important cellular processes (cell growth, cell cycle, malignant transformation). This suggests that PAI-1, in addition to its well-known anticancer properties, plays an important role in cell signaling. We hope that by exploring PAI-1's structure and function we might be able to understand and separate the different effects of PAI-1 on cancer cells and develop more effective therapeutic strategies in cancer treatment.

Formulation and characterization of EGCG for the treatment of superficial bladder cancer.

In the United States, the annual incidence of bladder cancer is approximately 70,000 new cases, with a mortality rate of approximately 15,000/year. The most common subtype (70%) of bladder cancer is superficial, namely hte non-muscle invasive disease form limited to the urothelium. The rate of progression and recurrence is up to 40 and 70%, respectively. Urothelial cell carcinoma of the bladder is typically treated with transurethral resection. The cancerous cells can float onto the adjacent epithelium, increasing the risk of recurrence. The standard of care is to offer adjuvant intravesical agents to reduce the risk of progression and recurrence. Current intravesical treatments are costly and are associated with special biohazard handling protocols. Patients are treated with intravesical therapy with bacillus Calmetter­Guerin (BCG) bacterium, or mitomycin C (MMC) following resection, both of which can cause moderate to severe side-effects which are rarely life-threatening. We previously examined the efficacy of epigallocatechin-3-gallate (EGCG) in comparison with MMC to prevent tumor cell implantation/growth in an animal model of superficial bladder cancer. Experiments revile that EGCG is slightly more effective than MMC at decreasing tumor cell implantation and consequent cancer growth in a bladder. This treatment requires the stringent sterile requirement of EGCG. EGCG can be unstable when sterilized at high temperatures. Thus, we evaluated two low temperature sterilization methods, such as ionizing radiation or the filtration method followed by freeze-drying. Both methods ensure the sterility of the sample; however, infrared and HPLC analysis revealed a slightly better stability of irradiated EGCG over the filtration method. The concentration of stable free radicals following irradiation was low, which are unlikely to exert any damaging effects to EGCG. Therefore, we consider that radiation will be the preferred method of EGCG sterilization, and that this may prove useful for the effective use of EGCG in the treatment of bladder cancer.

In vivo and in vitro effect of baicalein on human prostate cancer cells.

We investigated the in vitro effects of baicalein and baicalin on human umbilical vein endothelial cells (HUVECs) and on human prostate tumor cells (DU-145 and PC3) as well as the effect of orally administered baicalein on the growth of DU-145 cells after subcutaneous injection into SCID mice. In vitro effects of baicalein and baicalin treatment on human prostate cancer cell lines DU-145 and PC-3 were assessed by employing cell proliferation (MTS) assay, cytotoxicity (LIVE/DEAD) assay, and TUNEL assay. In vitro anti-proliferative and anti-angiogenic properties of baicalein and baicalin were studied on HUVECs by sprout assay. The effect of orally administered baicalein on tumor growth in SCID mice was studied in four groups (n=10) of animals injected subcutaneously with DU-145 cells and treated daily for 28 days. The control group received only vehicle (carboxymethylcellulose), whereas the other three groups received escalating doses of baicalein (10, 20, and 40 mg/kg per day). Baicalein and baicalin exhibit dose-dependent growth inhibitory effects on human prostate cancer cells and umbilical vein endothelial cells in vitro. Also, treatment by these two flavonoid compounds significantly decreased the average number and length of sprouts formed by the endothelial cell aggregates in a dose-dependent manner. In vivo, treatment of mice with baicalein demonstrated a statistically significant tumor volume reduction (p<0.01) when compared to the control. This is the first study demonstrating an in vivo growth inhibitory effect of orally administered baicalein on human prostate tumors in mice.

Transperineal in vivo fluence-rate dosimetry in the canine prostate during SnET2-mediated PDT.

Advances in photodynamic therapy (PDT) treatment for prostate cancer can be achieved either by improving selectivity of the photosensitizer towards prostate gland tissue or improving the dosimetry by means of individualized treatment planning using currently available photosensitizers. The latter approach requires the ability to measure, among other parameters, the fluence rate at different positions within the prostate and the ability to derive the tissue optical properties. Here fibre optic probes are presented capable of measuring the fluence rate throughout large tissue volumes and a method to derive the tissue optical properties for different volumes of the prostate. The responsivity of the sensors is sufficient to detect a fluence rate of 0.1 mW cm(-2). The effective attenuation coefficient in the canine prostate at 660 nm is higher at the capsule (2.15+/-0.19 cm(-1)) than in proximity of the urethra (1.84+/-0.36 cm(-1)). Significant spatial and temporal intra- and inter-canine variability in the tissue optical properties was noted, highlighting the need for individualized monitoring of the fluence rate for improved dosimetry.

Epigallocatechin-3-gallate prevents tumor cell implantation/growth in an experimental rat bladder tumor model.

The aim of this study was to determine the efficacy of epigallocatechin-3-gallate (EGCG) (Polyphenon E®) in comparison with mitomycin C (MMC) to prevent tumor cell implantation/growth in an animal model of superficial bladder cancer and search for possible mechanism(s) of action. Female Fisher 344 rats were used to study the effects of EGCG and mitomycin C for the prevention of transitional cell tumor implantation (AY-27). Twenty rats served as a control, tumor implantation and saline wash only. Sixty rats were treated with EGCG (100, 200 and 400 µM) intravesically for 60 or 120 min after tumor implantation. Thirty other rats were divided equally and pretreated with 400 µM EGCG or saline for 120 min before tumor initiation. In a separate series of experiments, 30 rats were treated 2 weeks after tumor initiation with saline or EGCG (400 µM). In a different experiment 39 rats were treated with: saline (n=10) EGCG (n=9) 400 µM, MMC (n=10) 0.5 µM, MMC (n=10) 400 µM. Rats were sacrificed 3 weeks following treatment. Gross and histological analyses were performed on the bladders. EGCG and mitomycin C prevented intravesical tumor growth in a concentration- and time-dependent manner. EGCG pretreatment or treatment 2 weeks post tumor implantation did not have therapeutic effects. Molecular modeling suggests that EGCG inhibits urokinase and matrix metalloproteinase-9. EGCG prevents intravesical tumor implantation/growth with a slightly better efficacy than mitomycin C in this experimental model. The data suggest that EGCG lowers proteolytic activity and lowers probability of cancer cell implantation rather than direct cancer cell killing.

Replacement of intestinal mucosa with urothelium in rat augmented bladders using intravesical photodynamic therapy with 5-aminolaevulinic acid.

PURPOSE: We evaluated the efficacy of intravesical aminolevulinic acid (delta-aminolevulinic acid hydrochloride) (Frontier Scientific, Logan, Utah) and photodynamic therapy for the removal of small intestinal mucosa in augmented bladders in a rat model. MATERIALS AND METHODS: Enterocystoplasty was performed in 70 female rats using a patch of terminal ileum. A total of 28 were used to determine the pharmacokinetics (0.3, 0.6 and 0.9 M) and dwell time (30, 60 and 90 minutes) of intravesically administered aminolevulinic acid to optimize intestinal mucosal absorption and minimize bladder mucosal absorption. The remaining augmented rats were treated with intravesical photodynamic therapy at light doses of 75, 100 and 125 J. Ileal and bladder tissues were evaluated by light microscopy. Cystometric studies to evaluate bladder volume were measured before and after photodynamic therapy. RESULTS: The concentration of 0.3 M aminolevulinic acid with a dwell time of 30 minutes resulted in an average +/- SE bowel-to-bladder concentration of 2,156 +/- 269/749 +/- 62 ng/gm (ratio 2.9:1). After photodynamic therapy histology revealed uniform ablation and replacement of the intestinal mucosa with urothelium and minimal damage to the bladder wall at all light doses. Bladder cystometry revealed no significant change in bladder capacity after photodynamic therapy. CONCLUSIONS: In the rat model intravesical aminolevulinic acid and photodynamic therapy resulted in the replacement of intestinal mucosa with urothelium, leaving the underlying muscular layer intact. This could potentially be a viable option for patients with a preexisting bladder augment.

Intraperitoneal chemotherapy for the prevention of transitional cell carcinoma implantation.

PURPOSE: Tumor spillage from bladder perforation during transurethral resection of a bladder tumor or during cystectomy risks seeding the peritoneum with TCC. Current therapy is irrigation with sterile water with an unknown extent of clinical benefit. Intraperitoneal chemotherapy for other human cancers has demonstrable benefit but to our knowledge it has never been investigated for TCC. We investigated whether intraperitoneal chemotherapy can prevent TCC implantation in a murine model of tumor spillage and whether water irrigation is beneficial. MATERIALS AND METHODS: Laparotomy was performed in 28 Fischer 344 rats (National Cancer Institute, Frederick, Maryland) to instill 1 x 10 AY-27 TCC cells. Mitomycin (10 mg/m) was instilled in 9 rats and saline was used in the control group. A third group underwent lavage with sterile water. At sacrifice after 2 weeks tumors were measured in mm and weighed. A followup experiment of 4-week survival used 5 mg/m mitomycin and added a fourth group treated with water lavage plus mitomycin. RESULTS: All 9 rats in the saline control group had gross tumors at the laparotomy site as well as gross carcinomatosis. The 10 water lavage rats also demonstrated gross tumors but of smaller size (p = 0.02). All rats treated with mitomycin had no gross or microscopic evidence of tumor growth anywhere in the peritoneum. In experiment 2 none of the rats treated with lower dose mitomycin had gross or microscopic tumors regardless of water lavage. CONCLUSIONS: Intraperitoneal chemotherapy prevents TCC implantation in a murine model of tumor spillage. Water lavage decreases the tumor burden but it cannot effectively sterilize the peritoneum of tumor.

Diverse optical characteristic of the prostate and light delivery system: implications for computer modelling of prostatic photodynamic therapy.

OBJECTIVE: To explore the use of photodynamic therapy (PDT) as a minimally invasive form of treatment for organ-confined prostate cancer, for although there are several therapies, ablative treatments are associated with significant morbidity. MATERIALS AND METHODS: Using the photosensitizer tin etiopurpurin, dogs were treated with interstitially placed laser fibres in an effort to validate PDT for treating prostate cancer. Earlier models assumed a uniform distribution of light output from a cylindrical fibre and a uniform attenuation coefficient throughout the prostate. Subsequent observations show that this model was too simple and that light radiance is not linear. To overcome under-treatment, a computer program to complement real-time fibre placement was developed. RESULTS: As light radiance from interstitially placed laser fibres varies significantly from the commonly assumed ideal cylindrical emission, a predictive mathematical model of prostate PDT needs to consider the real emission. Also, the optical properties of the prostate, e.g. absorption and scattering of light, are anisotropic. Differences in the attenuation coefficient (combining absorption and scattering of light) also varied among different animals. Incorporating all these variables into a computer program produced a virtual model of the photo-ablated zone within +/- 2 mm of that observed in animals. CONCLUSION: PDT of the prostate is not trivial and should benefit from computer-aided methods as it is developed for clinical use.

Inhibition of bladder tumor growth by the green tea derivative epigallocatechin-3-gallate.

PURPOSE: We evaluated the green tea derivative epigallocatechin-3-gallate (EGCG) as an intravesical agent for the prevention of transitional cell tumor implantation. MATERIALS AND METHODS: In vitro studies were performed in the AY-27 rat transitional cell cancer and the L1210 mouse leukemia cell lines. Cells were exposed to increasing concentrations of EGCG for 30 minutes to 48 hours. Surviving cell colonies were then determined. A DNA ladder assay was performed in the 2 cell lines. Fisher 344 rats were used for in vivo studies with an intravesical tumor implantation model. Group 1 (12 rats) served as a control (tumor implantation and medium wash only). In group 2 (28 rats) 200 microM EGCG were instilled intravesically 30 minutes after tumor implantation. Rats were sacrificed 3 weeks following treatment. Gross and histological analyses were then performed on the bladders. RESULTS: At 6.0 x 104 cells per 100 mm dish a time dose dependent response was observed. After 2 hours of treatment with EGCG 100% cell lethality of the AY-27 cell line occurred at concentrations greater than 100 microM. Strong banding on the DNA ladder assay was seen with the L1210 mouse leukemia cell line. Only weak banding patterns were found in the AY-27 cell line treated with EGCG (100 and 200 microM) for 24 hours. All 12 controls were successfully implanted with tumors. In group 2 (EGCG instillation) 18 of the 28 animals (64%) were free of tumor (Fisher's exact test p = 0.001). CONCLUSIONS: The clonal assays showed a time dose related response to EGCG. Intravesical instillation of EGCG inhibits the growth of AY-27 rat transitional cells implanted in this model.

Optical characteristics of the canine prostate at 665 nm sensitized with tin etiopurpurin dichloride: need for real-time monitoring of photodynamic therapy.

PURPOSE: Photodynamic therapy (PDT) is an emerging, minimally invasive therapy for prostate cancer that depends on the sequestration of a photosensitizing drug within targeted tissue. The photosensitizer is subsequently activated by light of a specific wavelength, resulting in destruction of the targeted tissue. Successful treatment requires knowledge of the optical properties of the target tissue, a critical element for therapy. MATERIALS AND METHODS: Adult canines were injected with tin etiopurpurin dichloride (1.0 mg/kg) as a liposome emulsion vehicle in saline 24 hours prior to light treatment. Laser light was delivered to the prostate via a 400 microm optical fiber fitted with a 2.0 cm cylindrical diffuser and optical properties of the prostate were measured. RESULTS: In this study we determined the attenuation coefficient and critical fluence in the canine prostate. Our studies shown that the attenuation coefficient is not uniform but higher at the base (average for all animals 2.59 to 2.79 cm-1) than in the mid section or apex of the prostate (1.71 to 1.90 cm-1). Significant differences among dogs (0.11 to 12.70 cm-1) were found. In some cases we observed a fluctuation of the attenuation coefficient during treatment. We also established experimentally the minimum energy (1449 mJ/cm2) needed (critical fluence) to produce necrosis. Experimentally establishing the values of effective attenuation and critical fluence is necessary to predict the area of ablation during PDT and protect surrounding organs from over treatment. CONCLUSIONS: Based on our results it is evident that for PDT of the prostate to be successful the optical parameters of the prostate must be measured and monitored during treatment. We suggest that the optimum way of doing this is real-time computerized monitoring combined with simulation PDT.

Use of small intestinal submucosa for corporal body grafting in cases of severe penile curvature.

PURPOSE: Small intestinal submucosa is a unique biomaterial that has been found to promote tissue specific regeneration in the urinary tract. We present our experimental and clinical experience with small intestinal submucosa (SurgiSis, Cook Biotech, Spencer, Indiana) for pediatric corporal body reconstruction. MATERIAL AND METHODS: A total of 20 Fischer rats underwent implantation of a 7 x 3 mm. small intestinal submucosa graft following excision of an ellipse of tunica albuginea and 14 control animals underwent tunical excision with reimplantation of this autologous segment. The animals were euthanized, and the penis was sectioned and histologically studied at intervals of 1, 2, 4, 6, 16 and 24 weeks. In 15 pediatric patients small intestinal submucosa was used for corporal body grafting. The grafting procedure was performed along the ventral (hypospadias cases) or dorsal (epispadias cases) surface of the corporal bodies. The tunica albuginea was incised full thickness at the point of maximal curvature down to the cavernosal tissue and the defect was filled with a single layer of small intestinal submucosa. RESULTS: Measurements of the animal small intestinal submucosa grafts did not reveal significant graft contraction through 6 months. There was no graft expansion or ballooning after pharmacological induction of an artificial erection. Histologically, marked inflammation at 1 week precipitously decreased to a normal appearing tunica albuginea at 3 and 6 months. In all clinical cases small intestinal submucosa was found to be technically easy to handle. Mean followup is 14 months (range 5 to 26). All patients have a straight phallus as documented by observation of spontaneous erections or artificial erection at the time of stage 2 hypospadias repair. No complications occurred. CONCLUSIONS: Small intestinal submucosa demonstrates tissue specific regeneration properties in the rat and human tunica albuginea. It is an off-the-shelf material that is safe, technically easy to use and readily available.