Growth inhibition of androgen-responsive prostate cancer cells with brefeldin A targeting cell cycle and androgen receptor.

BACKGROUND: Androgen ablation is one of the viable therapeutic options for patients with primary hormone (androgen)-dependent prostate cancer. However, an antibiotic brefeldin A (BFA) has been shown to exhibit the growth inhibitory effect on human cancer cells. We thus investigated if BFA might inhibit proliferation of androgen-responsive prostate cancer LNCaP cells and also explored how it would be carried out, focusing on cell cycle and androgen receptor (AR). METHODS: Androgen-mediated cellular events in LNCaP cells were induced using 5alpha-dihydrotestosterone (DHT) as an androgenic mediator. Effects of BFA on non-DHT-stimulated or DHT-stimulated cell growth were assessed. Its growth inhibitory mechanism(s) was further explored; performing cell cycle analysis on a flow cytometer, assessing AR activity by AR binding assay, and analyzing AR protein expression using Western blot analysis. RESULTS: DHT (1 nM) was capable of stimulating LNCaP cell growth by ~40% greater than non-stimulated controls, whereas BFA (30 ng/ml) completely inhibited such DHT-stimulated proliferation. Cell cycle analysis showed that this BFA-induced growth inhibition was associated with a ~75% reduction in the cell number in the S phase and a concomitant increase in the G1 cell number, indicating a G1 cell cycle arrest. This was further confirmed by the modulations of specific cell cycle regulators (CDK2, CDK4, cyclin D1, and p21WAF1), revealed by Western blots. In addition, the growth inhibition induced by BFA was accompanied by a profound (~90%) loss in AR activity, which would be presumably attributed to the significantly reduced cellular AR protein level. CONCLUSIONS: This study demonstrates that BFA has a potent growth inhibitory activity, capable of completely inhibiting DHT (androgen)-stimulated LNCaP proliferation. Such inhibitory action of BFA appears to target cell cycle and AR: BFA led to a G1 cell cycle arrest and the down-regulation of AR activity/expression, possibly accounting for its primary growth inhibitory mechanism. Thus, it is conceivable that BFA may provide a more effective therapeutic modality for patients with hormone-dependent prostate cancer.

Mode of cytotoxic action of nephrotoxic agents: oxidative stress and glutathione-dependent enzyme.

OBJECTIVE: To investigate the cytotoxic action of nephrotoxic agents using an in vitro renal cell model, focusing on the cellular oxidative status and a specific glutathione (GSH)-dependent enzyme, glyoxalase I (Gly-I). MATERIALS AND METHODS: Renal proximal tubular LLC-PK(1) cells were exposed to mercuric chloride, glycerol, cisplatin, gentamicin and cyclosporin A, and cell number/viability were determined. Oxidative stress was assessed by lipid peroxidation (LPO) assay, and Gly-I activity was measured by enzymatic method on a spectrophotometer. RESULTS: Both mercuric chloride (30 microm) and glycerol (2.5%) were highly toxic to LLC-PK(1) cells, inducing >90% cell death within 24 h. The remaining agents led to slightly >50% growth inhibition at 72 h. The LPO levels at 3 h in cells exposed to mercuric chloride or glycerol were approximately 2.5 times higher than that in controls. N-acetylcysteine (NAC), a potent antioxidant and precursor for GSH, almost completely (>95%) prevented renal cell death from mercuric chloride or glycerol. Gly-I activity was dependent on NAC and closely associated with cell viability. A approximately 65% loss in Gly-I activity by mercuric chloride/glycerol led to >90% cell death, while restoring a basal activity of Gly-I with NAC was accompanied by complete cell viability. CONCLUSIONS: The cytotoxic action of nephrotoxic agents appears to be triggered by oxidative stress, leading to Gly-I inactivation. As Gly-I plays a key role in cellular detoxification, its inactivation under oxidative stress probably becomes fatal to cells. However, cytoprotection provided with NAC is significant and might have implications in preventing renal cell injury mediated through nephrotoxic agents.

Synergistic potentiation of interferon activity with maitake mushroom d-fraction on bladder cancer cells.

OBJECTIVE: To examine whether the combination of interferon (IFN)-alpha and maitake mushroom D-fraction (PDF), a bioactive mushroom extract, might potentiate the anticancer activity of IFN-alpha in bladder cancer T24 cells in vitro. MATERIALS AND METHODS: Effects of recombinant IFN-alpha(2b) (0-50 000 IU/mL), PDF (0-700 microg/mL), or their combinations were assessed on T24 cell growth at 72 h. Cell cycle analysis and assays for double-stranded DNA-dependent protein kinase (DNA-PK) were performed to explore possible antiproliferative mechanism of these agents. RESULTS: IFN-alpha(2b) was able to induce a significant ( approximately 50%) growth reduction at 20 000 IU/mL, which further declined to approximately 66% at 50 000 IU/mL. PDF had no effects up to 200 microg/mL, but there was an approximately 20% and approximately 53% growth reduction at 400 and 700 microg/mL, respectively. When the varying concentrations of IFN-alpha(2b) and PDF were combined, 10 000 IU/mL of IFN-alpha(2b) combined with 200 microg/mL of PDF resulted in an approximately 75% growth reduction. This was accompanied by a G(1) cell cycle arrest, shown by cell cycle analysis. Concurrently, DNA-PK activity in IFN-alpha(2b)/PDF-treated cells was almost three-fold higher than controls. CONCLUSIONS: The combination of IFN-alpha(2b) (10 000 IU/mL) and PDF (200 microg/mL) reduced growth by approximately 75% in T24 cells. This appears to be due to a synergistic potentiation of these two agents, inducing a G(1) arrest with DNA-PK activation. Therefore, the IFN-alpha(2b)/PDF combination could trigger DNA-PK activation that may act on the cell cycle to cease cancer cell growth.

SX-fraction: Promise for novel treatment of type 2 diabetes.

SX-fraction (SXF) is a bioactive glycoprotein with anti-diabetic and hypoglycemic activities that have been documented in several reports. We have reviewed those studies herein and also explored the possible mechanism of its hypoglycemic activity. The early animal studies of SXF using diabetic mice showed the significant reduction in the three diabetic parameters, serum glucose, insulin, and triglyceride, suggesting its anti-diabetic activity. The limited clinical studies also showed that SXF led to the significant reduction in the fasting blood glucose levels of type 2 diabetic patients within 2 wk or a month, suggesting its hypoglycemic activity. To explore the hypoglycemic mechanism of SXF, its possible effects on the insulin signal transduction pathway was examined in vitro. Particularly, activities of insulin receptor, insulin receptor substrate 1, and protein kinase B, which are essential elements playing a key regulatory role in the signal pathway, were studied using skeletal muscle L6 cells. The status of these three parameters were examined under a high glucose (35 mmol/L) milieu with SXF and assessed using the enzyme-linked immunosorbent assay. Such studies revealed that all three parameters (insulin receptor, insulin receptor substrate 1, and protein kinase B) were inactivated by high glucose, indicating a disruption of the signal pathway. However, such an inactivation was reversed or re-activated by SXF to successfully carry out the sequential signaling events. In fact, a measurement of glucose uptake in cells showed that SXF did increase a glucose uptake while high glucose decreased it. Therefore, SXF has anti-diabetic and hypoglycemic activities through activation of the insulin signal pathway and appears to be a safe, natural agent for lowering the serum glucose levels in type 2 diabetic patents and improving their diabetic conditions.

Prophylactic effect of ethyl pyruvate on renal ischemia/reperfusion injury mediated through oxidative stress.

PURPOSE: As oxidative stress (OXS) has been shown to play a primary role in renal ischemia/reperfusion injury (RIRI), we investigated whether antioxidant such as ethyl pyruvate (EPy) might effectively prevent RIRI. Possible prophylactic effects of EPy and mannitol (Mann), one of perioperative agents often used, were tested against harmful OXS in vitro. METHODS: Hydrogen peroxide (H2O2) was used to exert OXS on the renal proximal tubular MDCK cells. Severity of OXS and protective effects of EPy and Mann were assessed by lipid peroxidation assay and cell viability test, respectively. The cytotoxic mechanism of H2O2 was explored by examining the status of glycolysis, metabolic signaling pathways, cell cycle, and induction of apoptosis. RESULTS: Although H2O2 (500 µM) increased OXS by ~ 3.5 times of controls, EPy (1 mM) fully reduced it to the basal level. Cell viability declined to merely 10% by H2O2 was regained to > 90% with EPy. Hexokinase activity and ATP level also declined significantly by H2O2, but they sustained 80-90% with EPy. Additionally, H2O2 led to the modulations of metabolic signaling regulators, a G1 cell cycle arrest, and induction of apoptosis, which were yet prevented with EPy. Unlike EPy, Mann had virtually little effects. CONCLUSIONS: OXS can indeed lead to the significant cell viability reduction through its adverse cellular effects, ultimately resulting in RIRI. However, EPy appears to prevent these effects and protect MDCK cells, while Mann does not. Thus, EPy could be a more effective prophylactic renoprotective agent (than Mann) against oxidative renal cell injury including RIRI.

Device Malfunctions and Complications Associated with Benign Prostatic Hyperplasia Surgery: Review of the Manufacturer and User Facility Device Experience Database.

Introduction and Objectives: Multiple surgical therapies for benign prostatic hyperplasia (BPH) have been developed to decrease complications and increase provider efficiency. We investigated contemporary BPH treatment device-related adverse events by searching a publicly available database. Materials and Methods: The Manufacturer and User Facility Device Experience (MAUDE) database was queried for contemporary BPH treatments. All devices were evaluated for malfunction, patient complications, and manufacturer review. The MAUDE adverse event classification system was used to standardize complications. Univariate analysis was performed to identify associations between BPH devices and adverse events. Results: A total of 2567 reports were identified: transurethral resection of the prostate (TURP) 197 (7.67%), holmium laser enucleation of the prostate (HoLEP) 39 (1.52%), GreenLight™ 2315 (90.2%), and UroLift® 16 (0.62%). The most common deviations for each modality included cutting loop detachment during TURP 116 (58.9%), morcellator dysfunction for HoLEP 23 (58.9%), tip fracture/detachment for GreenLight (68.8%), and failure to deploy during UroLift 10 (62.5%). Only 18 (0.7%) patients required medical/surgical management (MAUDE II-IV) due to a device complication. No significant relationship was seen between each modality and complications; however, morcellator use (27.8%) was observed in higher grade complications. Manufacturer review occurred in 61.7% of cases, with 41.3% of reviewed cases finding the operator the cause of the malfunction. Conclusion: Each BPH modality investigated had minimal patient harm with over 99% of patients experiencing no complication after device malfunction. Of note, great care should be taken with morcellator use during HoLEP as it had the greatest number of MAUDE II to IV complications among all devices. Manufacturer review revealed that over 40% of cases were due to misuse by the user. Therefore, urologists should select the modalities they are most familiar with to decrease patient harm and prevent device malfunctions.

Effect of various natural products on growth of bladder cancer cells: two promising mushroom extracts.

Despite the availability of several therapeutic options, a safer and more effective modality is urgently needed for treatment of bladder cancer. Specific immunotherapy is effective, but severe side effects limit its clinical use and underscore the need for unconventional therapies using less toxic substances. Many natural substances are touted for their medicinal aspects and side effect profiles, and some of these have been well characterized for their biological and medicinal properties. Accordingly, the effects on bladder cancer cells in vitro were investigated. Eight commercially available natural products were tested for possible effects on the growth of human bladder cancer T24 cells. This study demonstrated that two mushroom extracts, GD- and PL-fractions, induced a significant (>90 percent) growth reduction in 72 hours, whereas the remaining six products had no effect. Interestingly, non-toxic concentrations of the GD- or PL-fractions, when combined with a non-toxic concentration of vitamin C, became highly cytotoxic, resulting in >90-percent cell death. Thus, vitamin C appears to act synergistically with these fractions to potentiate their bioactivity (cytotoxicity). No other products tested demonstrated such a synergistic potentiation with vitamin C. The present study indicates that GD- and PL-fractions appear to have the most potent cytotoxic effect on human bladder cancer T24 cells. It is thus plausible that these substances could be used, solely or combined with conventional modalities, for the treatment of superficial bladder cancer.

Device-Related Adverse Events During Percutaneous Nephrolithotomy: Review of the Manufacturer and User Facility Device Experience Database.

INTRODUCTION AND OBJECTIVES: Percutaneous nephrolithotomy (PCNL) is an established technique for removal of large stones from the upper urinary tract. It is a complex multistep procedure requiring several classes of instruments that are subject to operator misuse and device malfunction. We report device-related adverse events during PCNL from the Manufacturer and User Facility Device Experience (MAUDE) database using a recently developed standardized classification system. MATERIALS AND METHODS: The MAUDE database was queried for "percutaneous nephrolithotomy" from 2006 to 2016. The circumstances and patient complications associated with classes of devices used during PCNL were identified. We then utilized a novel MAUDE classification system to categorize clinical events. Logistic regression analysis was performed to identify associations between device classes and severe adverse events. RESULTS: A total of 218 device-related events were reported. The most common classes included: lithotripter 53 (24.3%), wires 43 (19.7%), balloon dilators 30 (13.8%), and occlusion balloons 28 (12.8%). Reported patient complications included need for a second procedure 12 (28.6%), bleeding 8 (19.0%), retained fragments 7 (16.7%), prolonged procedure 4 (9.5%), ureteral injury 2 (4.8%), and conversion to an open procedure 3 (7.1%). Using a MAUDE classification system, 176 complications (81%) were Level I (mild/none), 26 (12%) were Level II (moderate), 15 (7%) were Level III (severe), and 1 (0.5%) was Level IV (life threatening). On univariate analysis, balloon dilators had the highest risk of Level II-IV complications compared with the other device classes [odds ratio: 4.33, confidence interval: 1.978, 9.493, p < 0.001]. The device was evaluated by the manufacturer in 93 (42.7%) cases, with 54.8% of reviewed cases listing the source of malfunction as misuse by the operator. CONCLUSIONS: PCNL is subject to a wide range of device-related adverse events. A MAUDE classification system is useful for standardized, clinically-relevant reporting of events. Our findings highlight the importance of proper surgeon training with devices to maximize efficiency and decrease harm.

Antioxidant and Renoprotective Effects of Mushroom Extract: Implication in Prevention of Nephrolithiasis.

BACKGROUND: The pathogenesis of nephrolithiasis (kidney stone) remains elusive, while several therapeutic options are available but not effective as we expected. Accumulating data yet suggest that oxidative stress (generation of oxygen free radicals) may play a primary role in its occurrence. Particularly, calcium oxalate (CaOx) is a key element in the most common form (> 75%) of kidney stones, and its crystal form known as CaOx monohydrate (COM) has been shown to exert oxidative stress, facilitating CaOx stone formation. Hence, diminishing oxidative stress with certain antioxidants could be a potential strategic approach. We are interested in a bioactive extract of Poria mushroom, PE, which has been shown to have antioxidant and renoprotective activities. Accordingly, we investigated if PE might have antioxidant activity that would have implication in prevention of kidney stone formation. METHODS: Renal epithelial LLC-PK1 cells were employed and exposed to COM or hydrogen peroxide (H2O2) as a positive control capable of exerting oxidative stress. Possible antioxidant and protective effects of PE against oxidative stress (exerted by COM or H2O2) were assessed by cell viability test and lipid peroxidation (LPO) assay. To explore its protective mechanism, two glycolytic parameters, hexokinase (HK) activity and ATP synthesis, were examined and cell cycle analysis was also performed. RESULTS: Both H2O2 and COM led to a significant (P < 0.05) reduction in cell viability, accompanied by severe oxidative stress assessed by LPO assay. Such oxidative stress also caused the significant decline in HK activity and cellular ATP level, indicating the inhibition of glycolysis. Cell cycle analysis further indicated that oxidative stress interfered with cell cycle, inducing a G1 cell cycle arrest that presumably results in the cessation of cell proliferation. However, PE was capable of significantly preventing or diminishing all these cellular effects mediated through oxidative stress (exerted by H2O2 and COM). CONCLUSIONS: The present study shows that the mushroom extract PE appears to have antioxidant and renoprotective effects against oxidative stress exerted by COM in renal cells. Therefore, PE with antioxidant activity is considered a promising natural agent that may have clinical implications in prevention of nephrolithiasis primarily induced by oxidative stress.

Potent Anticancer Effects of Bioactive Mushroom Extracts (Phellinus linteus) on a Variety of Human Cancer Cells.

BACKGROUND: Although several therapeutic options are currently available for patients with various cancers, the outcomes are often disappointing and a more effective modality needs to be promptly established. We have been exploring an alternative approach using natural agents and two bioactive mushroom extracts isolated from Phellinus linteus (PL), namely PL-ES and PL-I-ES, were of our interest. As anticancer effects of similar extracts have been reported in several cancers, we investigated whether PL-ES and PL-I-ES might have such anticancer activities on a variety of human cancer cells in vitro. METHODS: Ten different types of human cancer cell lines, including three metastatic prostate, bladder, kidney, lung, breast, stomach, liver, and brain cancer cells, were employed and tested with PL-ES or PL-I-ES. Cell growth/viability, exertion of oxidative stress, and induction of apoptosis were assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay, lipid peroxidation (LPO) assay, and specific enzymatic assay, respectively. RESULTS: PL-ES (100 µg/mL) exhibited potent anticancer activity, resulting in a significant (40-80%) growth reduction in all 10 cancer cells at 72 hours. PL-I-ES (100 µg/mL) was effective on only four cancer cells but its higher concentration at 250 µg/mL led to a significant (25-90%) growth reduction in seven cancer cells. LPO assays indicated that such a significant growth reduction by PL-ES (100 µg/mL) or PL-I-ES (100 or 250 µg/mL) could result from cell death due to a cytotoxic effect of oxidative stress (through free radicals). Moreover, enzymatic assays for caspase-3 (Csp-3) and caspase-9 (Csp-9), the pro-apoptotic regulators, showed that both enzymes were significantly activated by PL-ES or PL-I-ES, indicating that cell death due to oxidative stress was more likely associated with apoptosis. CONCLUSIONS: The present study shows that both PL-ES and PL-I-ES indeed have anticancer effects on a variety of cancer cells, although PL-ES appears to be more potent than PL-I-ES. Such an anticancer effect is presumably attributed to oxidative stress, which will ultimately lead to apoptosis. Therefore, these two bioactive mushroom extracts may have clinical implications in a more effective therapeutic option for a variety of human malignancies.

Salvage extracorporeal shockwave lithotripsy after failed distal ureteroscopy.

BACKGROUND AND PURPOSE: When intervention is necessary, controversy remains as to the best treatment modality for stones of the distal ureter. In general, ureteroscopy is favored over extracorporeal shockwave lithotripsy (SWL) as the treatment of choice for distal ureteral stones. Although uncommon, ureteroscopy failures have traditionally necessitated repeat ureteroscopy to retrieve retained stone fragments. We evaluated the efficacy of salvage SWL for failed primary distal ureteroscopy in the community setting. PATIENTS AND METHODS: From December 1989 to December 2000, 6099 patients underwent SWL with the Dornier HM4 lithotripter at our institution. We retrospectively identified 31 patients who had undergone the SWL after a failed distal ureteroscopy. RESULTS: The average stone size in these patients was 9.4 mm, the average time interval from ureteroscopy to SWL was 17.2 days, and the average number of shockwaves delivered was 2386. All patients had had stents placed after ureteroscopy. Twenty-seven patients (87%) had resolution of their stone burden after one SWL session. The remaining four patients underwent additional procedures. CONCLUSIONS: Ureteroscopy is an effective modality for the treatment of distal ureteral stones. However, when unsuccessful, a salvage procedure may be necessary. Extracorporeal lithotripsy is a less invasive procedure with comparable success rates in the distal ureter. This report suggests that salvage SWL is an appropriate option for patients in whom distal ureteroscopic stone extraction fails.

Chemosensitization of carmustine with maitake beta-glucan on androgen-independent prostatic cancer cells: involvement of glyoxalase I.

OBJECTIVE: To improve the poor efficacy (< 10%) of chemotherapy for patients with hormone-refractory prostate cancer, we investigated a possible cytotoxic effect of carmustine/beta-glucan combination on prostatic cancer PC-3 cells, focusing on a glutathione-dependent detoxifying enzyme, glyoxalase I (Gly-I). METHODS: Carmustine (BCNU) is an anticancer agent and a putative inhibitor of Gly-I, while beta-glucan is a unique, nontoxic polysaccharide extracted from maitake mushrooms. The cytotoxic effects of BCNU or other anticancer agents with beta-glucan on PC-3 cells were assessed by cell-viability testing and Gly-I activity was measured using the spectrophotometric method. RESULTS: BCNU, 5-fluorouracil (5-FU), and methotrexate (MTX) were capable of inducing approximately a 50% reduction in cell viability at 72 hours, while etoposide, cisplatin, and mitomycin C were all ineffective. Only the combination of BCNU (50 micro ;mol) and beta-glucan (60 micro g/mL) exhibited an enhanced cytotoxicity with an approximate 90% cell viability reduction, but little improvement was seen with any combinations of 5-FU, MTX, or beta-glucon. Gly-I assays revealed that such a profound (approximately 90%) cell death was accompanied by an approximate 80% reduction in Gly-I activity by 6 hours. CONCLUSION: This study demonstrates a sensitized cytotoxic effect of BCNU with beta-glucan in PC-3 cells, which was associated with a drastic (approximately 80%) inactivation of Gly-I. Therefore, the BCNU/beta-glucan combination may help to improve current treatment efficacy by targeting Gly-I, which appears to be critically involved in prostate cancer viability.

Potential growth inhibitory effect of maitake D-fraction on canine cancer cells.

The postulated anticancer effect of D-fraction, the bioactive extract of maitake mushroom, on three types (CF33, CF21, and CL-1) of canine cancer cells was evaluated. The effect of D-fraction on several human cancer cells was also investigated. The effect of other beta-glucan products was likewise examined. D-fraction was highly effective on the canine cancer cells, either potently inhibiting cell growth or directly killing cells. Similar effects were also demonstrated in certain human cancer cells. However, other beta-glucan products relevant to D-fraction had no such effects on canine cancer cells. Therefore, D-fraction is a potent natural agent that could be useful in treating canine cancers as well as other veterinary cancers.

Alternative therapeutic approach to renal-cell carcinoma: induction of apoptosis with combination of vitamin K3 and D-fraction.

PURPOSE: Because of a dismal prognosis for advanced renal-cell carcinoma (RCC), an alternative therapeutic approach, using vitamin K3 (VK3) and D-fraction (DF) was investigated. VK3 is a synthetic VK derivative and DF is a bioactive mushroom extract, and they have been shown to have antitumor activity. We examined if the combination of VK3 and DF would exhibit the improved anticancer effect on RCC in vitro. MATERIALS AND METHODS: Human RCC, ACHN cell line, were treated with varying concentrations of VK3, DF, or a combination of the two. Cell viability was assessed at 72 hours by MTT assay. To explore the possible anticancer mechanism, studies on cell cycle, chromatin modifications, and apoptosis were conducted. RESULTS: VK3 alone led to a ~20% reduction in cell viability at 4 µM, while DF alone induced a 20% to 45% viability reduction at ≥ 500 µg/mL. A combination of VK3 (4 µM) and DF (300 µg/mL) led to a drastic >90% viability reduction, however. Cell cycle analysis indicated that VK3/DF treatment induced a G1 cell cycle arrest, accompanied by the up-regulation of p21(WAF1) and p27(Kip1). Histone deacetylase (HDAC) was also significantly (~60%) inactivated, indicating chromatin modifications. In addition, Western blot analysis revealed that the up-regulation of Bax and activation of poly-(ADP-ribose)-polymerase (PARP) were seen in VK3/DF-treated cells, indicating induction of apoptosis. CONCLUSIONS: The combination of VK3 and DF can lead to a profound reduction in ACHN cell viability, through a p21(WAF1)-mediated G1 cell cycle arrest, and ultimately induces apoptosis. Therefore, the combination of VK3/DF may have clinical implications as an alternative, improved therapeutic modality for advanced RCC.

Possible hypoglycemic action of SX-fraction targeting insulin signal transduction pathway.

BACKGROUND: SX-fraction (SXF) is a bioactive glycoprotein with hypoglycemic activity that has been demonstrated in our pilot clinical study. However, how it would actually work in diabetic patients remains unclear. To explore such a mechanism, the effects of SXF on the insulin signal transduction pathway were investigated using skeletal muscle L6 cells in vitro. METHODS: L6 cells were first differentiated to myotubes expressing several biochemical parameters that were examined in this study. Myotubes were exposed to a high concentration (35 mM) of glucose (Glc) alone or in combination with SXF or insulin for 24 hours. Possible effects of these agents on activities of insulin receptor (IR), IR substrate 1 (IRS-1), and Akt, which are key elements involved in the signal pathway, were assessed using enzyme-linked immunosorbent assay (ELISA). Any changes in Glc uptake were also determined. RESULTS: High Glc indeed led to inactivation of IR, IRS-1, and subsequent Akt in myotubes, indicating an interruption of the signal pathway. However, such inactivation was reversed or reactivated by SXF, presumably aiding the occurrence of successive signaling events. Measurement of Glc uptake to assess the outcome of this signaling cascade showed that high Glc decreased Glc uptake (interfering with the signal pathway), but SXF was capable of overcoming such a suppressive effect, resulting in the increased Glc uptake. Insulin was used as a positive control in this study and all results were nearly compatible to those obtained from SXF. CONCLUSION: The present study suggests that SXF may specifically target the insulin signal pathway, and, in particular, the IR and IRS-1 therein that trigger the subsequent signaling events. As a result, SXF could activate such an impaired signal pathway through high Glc or under a hyperglycemic milieu, thereby ultimately facilitating Glc uptake. This may then account for possible hypoglycemic action of SXF.

Preventive effect of specific antioxidant on oxidative renal cell injury associated with renal crystal formation.

OBJECTIVE: To investigate whether calcium oxalate monohydrate (COM), a key element of hyperoxaluria, would induce renal cell injury through oxidative stress and also whether certain antioxidants could prevent chemically induced renal crystal formation in rats. MATERIALS AND METHODS: COM-exerted oxidative stress on the kidney epithelial Madin-Darby canine kidney cells was assessed using the lipid peroxidation assay. Glyoxalase I (Gly-I) activity was also determined. Two antioxidants, vitamin C and N-acetylcysteine (NAC), were then tested to determine whether they could abolish such oxidative stress in Madin-Darby canine kidney cells. Both antioxidants were also tested to determine whether they might prevent or reduce renal crystal formation induced with ethylene glycol (EG) and vitamin D3 (VD3) in Wistar rats. RESULTS: COM (200 µg/mL) demonstrated ∼1.3-fold greater oxidative stress with a significant reduction in cell viability and Gly-I activity compared with controls. However, such adverse events were almost completely prevented with NAC but not with vitamin C. In the animal study, no renal crystals were seen in the sham group. However, numerous crystals, with reduced Gly-I activity and elevated oxidative stress, were found in the EG-VD3 group. However, markedly (>70%) fewer crystals, with full Gly-I activity and diminished oxidative stress, were detected in the EG-VD3+NAC group. CONCLUSION: COM exerted oxidative stress on Madin-Darby canine kidney cells, leading to cell viability reduction and Gly-I inactivation, with NAC fully preventing such adverse consequences. Similarly, numerous crystals with Gly-I inactivation and elevated oxidative stress seen in the rats (EG-VD3) were also significantly prevented with NAC supplement. Thus, NAC might have clinical implications in preventing oxidative renal cell injury and, ultimately, kidney stone formation.

Attenuation of androgenic regulation by brefeldin A in androgen-responsive prostate cancer cells.

OBJECTIVES: To investigate the effects of an antibiotic brefeldin A (BFA) on androgen-regulated cellular events in androgen-responsive prostate cancer cells, focusing on PSA (prostate-specific antigen) status, cell growth, and bioactivity of androgen receptor (AR). MATERIALS AND METHODS: Androgen-responsive human prostate cancer LNCaP cells were employed and 5α-dihydrotestosterone (DHT) was used as an androgenic mediator to induce androgen-modulated cellular events. Effects of BFA on synthesis and secretion of PSA, cell growth, and AR activity were assessed using Tandem PSA assay, trypan blue exclusion method, and AR binding assay, respectively. RESULTS: BFA (30 ng/ml) dramatically (90%) blocked secretion of PSA and also reduced cell growth by >75% under non-androgen-regulated condition. Under androgen-stimulated condition using DHT (1 nM), both the cellular and secreted PSA levels as well as cell growth was significantly elevated or stimulated by DHT (compared with controls); however, BFA was capable of completely inhibiting such DHT-stimulated cellular events. In addition, AR binding assay revealed that AR activity has been drastically (~90%) diminished by BFA, likely resulting in interruption of DHT-mediated events. CONCLUSIONS: BFA is capable of attenuating androgenic regulation in LNCaP cells such as androgen-stimulated PSA synthesis/secretion and cell growth. This BFA-blocked androgen action appears to be primarily attributed to severe inactivation of AR with BFA because AR is a crucial factor for relaying androgenic messages (to DNA). Therefore, BFA could be considered a promising agent for a more effective treatment of hormone-dependent prostate cancer.

Induction of cell death in renal cell carcinoma with combination of D-fraction and vitamin C.

HYPOTHESIS: Although several conventional therapeutic options for advanced renal cell carcinoma (RCC) are currently available, the unsatisfactory outcomes demand establishing more effective interventions. D-fraction (PDF), a bioactive proteoglucan of Maitake mushroom, demonstrates anticancer and immunomodulatory activities, which are also shown to be potentiated by vitamin C (VC). We thus hypothesized that a combination of PDF and VC (PDF + VC) could be an alternative approach to more effectively inhibit the growth of RCC. STUDY DESIGN: We examined the dose-dependent effects of PDF + VC on RCC cell viability and also performed biochemical assays to explore the growth regulatory mechanism. METHODS: Human RCC, ACHN cell line, was employed and exposed to varying concentrations of PDF or VC and their combinations. Cell viability at specified times was determined by MTT assay. Lipid peroxidation assay, cell cycle analysis, and Western blot analysis were also performed. RESULTS: PDF or VC alone led to the significant reduction in cell viability at 72 hours with PDF >500 µg/mL and VC ≥300 µM. When various combinations of PDF and VC were tested, the combination of the ineffective concentrations of PDF (300 µg/mL) and VC (200 µM) resulted in ~90% cell death in 24 hours. Lipid peroxidation assay then indicated significantly (~2.5 fold) elevated oxidative stress with this PDF + VC. Cell cycle analysis also indicated a G1 cell cycle arrest following a 6-hour PDF + VC treatment. Western blots further revealed a downregulation of Bcl2, an upregulation of Bax, and proteolytic activation of PARP (poly[ADP-ribose] polymerase) in PDF + VC-treated cells, indicating induction of apoptosis. CONCLUSION: The present study demonstrates that the combination of PDF and VC can become highly cytotoxic, inducing severe cell death in ACHN cells. This cytotoxic mechanism appears to be primarily attributed to oxidative stress, accompanied by a G1 cell cycle arrest. Such cell death induced by PDF + VC could be more likely linked to apoptosis, as indicated by the modulation of apoptosis regulators (Bcl2, Bax, and PARP). Therefore, as PDF and VC may work synergistically to induce apoptotic cell death, they may have clinical implications in an alternative, improved therapeutic modality for advanced RCC.

Nephrotoxin-induced renal cell injury involving biochemical alterations and its prevention with antioxidant.

BACKGROUND: Although nephrotoxic agents or nephrotoxins are known to induce acute renal cell injury, their cytotoxic action is not fully elucidated. It is thus crucial to explore such a cytotoxic mechanism and the increasing volume of reports indicated a significant involvement of oxidative stress. To test this possibility, we investigated if a nephrotoxin would exert oxidative stress, leading to renal cell injury accompanied by certain biochemical alterations. We also examined if specific antioxidant might help prevent such oxidative cell injury. These studies may then help establish a prophylactic or preventive modality for renal cell injury induced by nephrotoxins. METHODS: As glycerol has been commonly used for studying acute renal failure in animals, whether it would induce cellular injury was tested in renal proximal tubular OK cells in vitro. Cells were exposed to the varying concentrations of glycerol and cell number/viability was determined in 24 hours. Severity of oxidative stress was assessed by lipid peroxidation assay. Possible effects of glycerol on biochemical parameters were also examined on glyoxalase I activity and heat shock protein 90 using spectrophotometric (enzymatic) assay and Western blot analysis. RESULTS: Glycerol (2.5%) was highly cytotoxic to OK cells, inducing 95% cell death in 24 hours. Lipid peroxidation assay indicated that nearly 3-fold greater oxidative stress was exerted by this glycerol. Concurrently, glyoxalase I activity was drastically lost by 75% and heat shock protein 90 was partially degraded following glycerol exposure. However, N-acetylcysteine, a potent glutathione-based antioxidant, was capable of almost completely preventing the glycerol-mediated adverse outcomes, such as cell death, glyoxalase I inactivation, and heat shock protein 90 degradation. CONCLUSIONS: Glycerol is cytotoxic, capable of inducing specific biochemical alterations such as inactivation of glyoxalase I and degradation of heat shock protein 90, which may reflect a breakdown of the cellular detoxification and defense systems, leading ultimately to OK cell death. Nevertheless, as N-acetylcysteine can provide full cytoprotection against such glycerol toxicity, it could be considered a prophylactic modality for nephrotoxin-induced oxidative renal cell injury and death. KEYWORDS: Glycerol; Glyoxalase I; Heat shock protein; N-acetylcysteine; Renal cell injury.

Additively enhanced antiproliferative effect of interferon combined with proanthocyanidin on bladder cancer cells.

Although interferon (IFN) has been often used as immunotherapy for bladder cancer, its efficacy is rather unsatisfactory, demanding further improvement. Combination therapy is one of viable options, and grape seed proanthocyanidin (GSP) could be such an agent to be used with IFN because it has been shown to have anticancer activity. We thus investigated whether combination of IFN and GSP might enhance the overall antiproliferative effect on bladder cancer cells in vitro. Human bladder cancer T24 cells were employed and treated with the varying concentrations of recombinant IFN-α(2b) (0-100,000 IU/ml), GSP (0-100 µg/ml), or their combinations. IFN-α(2b) alone led to a ~50% growth reduction at 20,000 (20K) IU/ml, which further declined to ~67% at ≥50K IU/ml. Similarly, GSP alone induced a ~35% and ~100% growth reduction at 25 and ≥50 µg/ml, respectively. When IFN-α(2b) and GSP were then combined, combination of 50K IU/ml IFN-α(2b) and 25 µg/ml GSP resulted in a drastic >95% growth reduction. Cell cycle analysis indicated that such an enhanced growth inhibition was accompanied by a G(1) cell cycle arrest. This was further confirmed by Western blot analysis revealing that expressions of G(1)-specific cell cycle regulators (CDK2, CDK4, cyclin E and p27/Kip1) were distinctly modulated with such IFN-α(2b)/GSP treatment. Therefore, these findings support the notion that combination of IFN-α(2b) and GSP is capable of additively enhancing antiproliferative effect on T24 cells with a G(1) cell cycle arrest, implying an adjuvant therapeutic modality for superficial bladder cancer.