Tumor cell-based liquid biopsy using high-throughput microfluidic enrichment of entire leukapheresis product.

Circulating Tumor Cells (CTCs), interrogated by sampling blood from patients with cancer, contain multiple analytes, including intact RNA, high molecular weight DNA, proteins, and metabolic markers. However, the clinical utility of tumor cell-based liquid biopsy has been limited since CTCs are very rare, and current technologies cannot process the blood volumes required to isolate a sufficient number of tumor cells for in-depth assays. We previously described a high-throughput microfluidic prototype utilizing high-flow channels and amplification of cell sorting forces through magnetic lenses. Here, we apply this technology to analyze patient-derived leukapheresis products, interrogating a mean blood volume of 5.83 liters from patients with metastatic cancer, with a median of 2,799 CTCs purified per patient. Isolation of many CTCs from individual patients enables characterization of their morphological and molecular heterogeneity, including cell and nuclear size and RNA expression. It also allows robust detection of gene copy number variation, a definitive cancer marker with potential diagnostic applications. High-volume microfluidic enrichment of CTCs constitutes a new dimension in liquid biopsies.

Comparison of sexual function after robot-assisted radical prostatectomy and carbon-ion radiotherapy for Japanese prostate cancer patients using propensity score matching.

BACKGROUND: The quality of life of patients is an important consideration when selecting treatments for localized prostate cancer (PCa). We retrospectively compared sexual function after robot-assisted radical prostatectomy (RARP) and carbon-ion radiotherapy (CIRT) using propensity score matching. METHODS: In total, 127 Japanese PCa patients treated with RARP and 190 treated with CIRT monotherapy were evaluated. We evaluated the Expanded Prostate Cancer Index Composite (EPIC) score before treatment and 12 and 24 months after treatment. After propensity score matching, data from 101 patients from each group were analyzed. The study protocol was approved by the Institutional Review Board of Gunma University Hospital (no. IRB2020-050, 1839). RESULTS: After propensity score matching, the mean EPIC sexual function summary scores in the RARP and CIRT groups were 46.4 and 48.2, respectively. At 12 and 24 months after treatment, these scores were 27.9 (39.9% decrease) and 28.2 (39.2% decrease) in the RARP group and 41.4 (14.1% decrease) and 41.6 (13.7% decrease) in the CIRT group, respectively. Both groups demonstrated significantly decreased scores after 12 and 24 months of treatment compared to before treatment (all p < 0.05). At 12 and 24 months, the sexual function summary score was significantly higher in the CIRT group than in the RARP group (p < 0.001). CONCLUSIONS: There was a smaller decrease in the EPIC sexual function score in the CIRT group than in the RARP group. These results provide useful information for treatment decision-making of Japanese PCa patients.

Simvastatin Enhances the Radiosensitivity of Androgen-independent Prostate Cancer Cells via Inhibition of RAD51 Expression.

BACKGROUND/AIM: Statins exert antitumor effects via various mechanisms. Additionally, the recurrence rate of prostate cancer after radiation therapy is lower in patients taking statins. This study investigated the efficacy of combination therapy with statins and irradiation in androgen-independent prostate cancer cells. MATERIALS AND METHODS: PC-3 and LNCaP human prostate cancer cell lines were used in this study. We developed androgen-independent LNCaP cells (LNCaP-LA) by gradually replacing fetal bovine serum (FBS) with charcoal-stripped FBS. Microarray analysis was performed, followed by Ingenuity Pathway Analysis. Cell viability was determined using the MTS assay. RESULTS: Simvastatin alters gene expressions in PC-3 cells. Microarray data showed that the number of differentially expressed genes was the highest in the pathway of "Role of BRCA1 in DNA Damage Response". In the validation, the expression of RAD51, listed in "Role of BRCA1 in DNA Damage Response", decreased significantly by simvastatin in PC-3 cells. Reduction in RAD51 expression following siRNA transfection increased the cytocidal effects of X-ray therapy in PC-3 and LNCaP-LA cells. The combination of simvastatin and irradiation further inhibited cell proliferation compared with monotherapy with either therapy in PC-3 or LNCaP-LA cells. In addition, compared with X-ray monotherapy, the combination of simvastatin and irradiation further enhanced the expression of γH2AX, which is reported to be one of the accurate markers of DNA damage in PC-3 cells. CONCLUSION: Simvastatin decreased the expression of RAD51 in androgen-independent prostate cancer cells. The combination of irradiation and drugs that reduce RAD51 expression can potentially affect androgen-independent prostate cancer growth.

Simvastatin Induces Autophagy and Inhibits Proliferation in Prostate Cancer Cells.

BACKGROUND/AIM: Statin has recently been studied for its effects on inducing cell death and inhibiting metastasis. Nevertheless, the precise mechanism of its anti-tumor effect is not yet fully understood. We conducted research on statin as a novel treatment for castration-resistant prostate cancer (CRPC). This study focused on autophagy in prostate cancer cells and assessed the effects of simvastatin. MATERIALS AND METHODS: After administering simvastatin to PC-3 cells, we conducted a microarray analysis. Simvastatin was administered to prostate cancer cell lines (PC-3, LNCaP-LA; cultured under androgen-depleted conditions, DU145, 22RV1), and the tumor proliferation inhibition was evaluated using the MTS assay and cell count. Autophagy was measured by observing autophagosome staining under a fluorescence microscope and quantifying LC-3 protein using western blot. We also investigated the effects of rapamycin, an autophagy inducer, and chloroquine as an inhibitor. RESULTS: Simvastatin demonstrated a significant concentration-dependent growth inhibition effect on prostate cell lines. Moreover, a significant increase in autophagy was observed in all cell lines following simvastatin administration. When we administered simvastatin with rapamycin at a concentration that did not show a tumor growth inhibitory effect, it significantly enhanced autophagy induction compared to simvastatin alone, and also significantly enhanced the growth inhibition effect on PC-3 cells. CONCLUSION: Simvastatin induced autophagy and inhibited the proliferation of prostate cancer cell lines. The combination of simvastatin and rapamycin significantly induced autophagy and enhanced the inhibitory effect of simvastatin on proliferation. This mechanism may serve as a novel therapeutic target.

Predictive models of long-term survival outcomes following radical cystectomy.

BACKGROUND: Identifying the likelihood of life-threatening recurrence after radical cystectomy by reliable and user-friendly predictive models remains an unmet need in the clinical management of invasive bladder cancer. METHODS: A total of 204 consecutive patients undergoing open radical cystectomy (ORC) for bladder cancer were retrospectively enrolled between May 2005 and August 2020. Clinicopathological and peri-ORC therapeutic data were extracted from clinical records. We explored predictive factors that significantly affected the primary endpoint of overall survival (OS) and secondary endpoints of cancer-specific survival (CSS) and recurrence-free survival (RFS). RESULTS: During a median follow-up of 3.9 years, 42 (20.6%) and 10 (4.9%) patients died due to bladder cancer and other causes, respectively. Five-year RFS, CSS, and OS were 66.5%, 77.6%, and 75.4%, respectively. Pathological T and N categories and lymphovascular invasion (LVI) significantly affected RFS by Cox regression analysis. Accordingly, clinical T and pathological N categories and LVI significantly affected CSS. Clinical T and pathological N categories, LVI, age, and ORC tumor grade significantly affected OS. Based on the assessment score for each independent risk factor, we developed the Gunma University Oncology Study Group (GUOSG) score, which predicts RFS, CSS, and OS. The GUOSG score classified four groups for RFS, three for CSS, and five for OS, with statistically significant distribution for nearly all comparisons. CONCLUSIONS: The GUOSG model is helpful to show individualized prognosis and functions as a risk-stratified historical cohort for assessing the lifelong efficacy of new salvage treatment regimens.

Blood Concentration of Cabazitaxel in a Patient Whose General Condition Worsened with Concomitant Use of Clarithromycin.

We encountered a case in which the general condition of a patient receiving cabazitaxel worsened with concomitant use of clarithromycin. Cabazitaxel is metabolized mainly by CYP3A4, and the frequency of adverse events is known to increase with increasing exposure. Although these drugs are not often quantified in daily practice, we quantified them because we considered it possible that the blood concentration of cabazitaxel had increased due to CYP3A4 inhibition of clarithromycin and that cabazitaxel-related adverse events had occurred. However, the concentration of cabazitaxel was not increased and we attributed the patient's deterioration to decreased tolerability of cabazitaxel. At least at a trough concentration of 70 ng/mL, which is the trough concentration when a normal dose of clarithromycin is administered, clarithromycin does not appear to have a significant effect on the blood concentration of cabazitaxel. This case suggests that the administration of the normal dose of clarithromycin might be relatively safe in patients receiving cabazitaxel.

Increasing age predicts adverse pathology including intraductal carcinoma of the prostate and cribriform patterns in deferred radical prostatectomy after upfront active surveillance for Gleason grade group 1 prostate cancer: analysis of prospective observational study cohort.

BACKGROUND: In men undergoing upfront active surveillance, predictors of adverse pathology in radical prostatectomy specimens, including intraductal carcinoma of the prostate and cribriform patterns, remain unknown. Therefore, we aimed to examine whether adverse pathology in radical prostatectomy specimens could be predicted using preoperative patient characteristics. METHODS: We re-reviewed available radical prostatectomy specimens from 1035 men prospectively enrolled in the PRIAS-JAPAN cohort between January 2010 and September 2020. We defined adverse pathology on radical prostatectomy specimens as Gleason grade group ≥3, pT stage ≥3, pN positivity or the presence of intraductal carcinoma of the prostate or cribriform patterns. We also examined the predictive factors associated with adverse pathology. RESULTS: All men analyzed had Gleason grade group 1 specimens at active surveillance enrolment. The incidence of adverse pathologies was 48.9% (with intraductal carcinoma of the prostate or cribriform patterns, 33.6%; without them, 15.3%). The addition of intraductal carcinoma of the prostate or cribriform patterns to the definition of adverse pathology increased the incidence by 10.9%. Patients showing adverse pathology with intraductal carcinoma of the prostate or cribriform patterns had lower biochemical recurrence-free survival (log-rank P = 0.0166). Increasing age at active surveillance enrolment and before radical prostatectomy was the only predictive factor for adverse pathology (odds ratio: 1.1, 95% confidence interval: 1.02-1.19, P = 0.0178; odds ratio: 1.12, 95% confidence interval: 1.02-1.22, P = 0.0126). CONCLUSIONS: Increasing age could be a predictive factor for adverse pathology. Our findings suggest that older men could potentially derive advantages from adhering to the examination schedule in active surveillance.

Retroperitoneal urothelial carcinoma arising after bladder diverticulectomy: a case report.

BACKGROUND: Urothelial carcinoma arises from the inner urothelial membrane of the renal pelvis, ureter, and bladder and often causes macrohematuria. Here, we report a rare case in which the patient developed non-symptomatic urothelial carcinoma anatomically outside the bladder wall 17 years after bladder diverticulectomy. CASE PRESENTATION: An 82-year-old male patient previously underwent gastrectomy for stomach cancer and partial hepatectomy for intrahepatic cholangiocarcinoma. Follow-up computed tomography revealed a tumor in the retroperitoneal space, where a bladder diverticulum was removed 17 years earlier. Multiparametric magnetic resonance imaging suggested that the tumor was malignant with rectal invasion. Subsequent computed tomography-guided percutaneous biopsy revealed that the tumor was urothelial carcinoma. The patient underwent two courses of neoadjuvant chemotherapy followed by pelvic exenteration with pelvic lymph node dissection. He is currently receiving adjuvant therapy with an immune checkpoint inhibitor and has had no recurrence for 3 months. CONCLUSIONS: Multiparametric magnetic resonance imaging is a helpful tool for predicting both tumor malignancy and invasion before a pathologically confirmed diagnosis. Although this case is rare, urologists should be aware of the occurrence of urothelial carcinoma after bladder diverticulectomy in cases of incomplete resection of the diverticulum.

The utility of the [-2]pro-prostate-specific antigen level as a prognostic marker in patients with castration-resistant prostate cancer treated with enzalutamide.

BACKGROUND: The prostate health index (phi) derived using [-2]pro-prostate-specific antigen (p2PSA), a precursor of PSA, has been shown to predict cancer in the gray zone. However, the utility of p2PSA in predicting outcomes for castration-resistant prostate cancer (CRPC) patients remains unknown. Therefore, in this study, we aimed to evaluate the usefulness of p2PSA in predicting the efficacy and prognosis of enzalutamide treatment in CRPC patients. METHODS: We conducted a prospective study of CRPC patients treated with enzalutamide at our institution, measuring p2PSA levels in 98 pre-treatment serum samples. All patients were divided into two groups based on the median values of each parameter. The PSA progression-free survival (PSA-PFS) and overall survival (OS) were compared using the Kaplan-Meier method. This study was approved by the Institutional Review Board of Gunma University Hospital (IRB No. 2021-092, 1983). RESULTS: The median PSA level before enzalutamide treatment was 25.59 ng/mL, the median p2PSA level was 208.75 pg/mL, and the median phi was 187.95. PSA, p2PSA, and phi were not all predictors of PSA-PFS. However, the OS was significantly better in the low-value groups (log-rank p-values of PSA, p2PSA, and phi were 0.024, 0.034, and 0.018, respectively). In the docetaxel (DOC)-naive group (n = 58), PSA was not a predictor of OS, but p2PSA and phi were significantly associated with better OS in the low group. This relationship was not observed in the DOC-treated group. CONCLUSIONS: Our study elucidates the usefulness of p2PSA in predicting outcomes for CRPC patients treated with enzalutamide. It suggests that p2PSA and phi may be prognostic markers after enzalutamide administration in CRPC patients.

A prospective study of the relationship between CRPC patient's quality of life after enzalutamide and serum testosterone levels measured via LC-MS/MS.

BACKGROUND: Enzalutamide (ENZ) is used in the treatment of patients with castration-resistant prostate cancer (CRPC). The quality of life (QoL) of CRPC patients during ENZ treatment is very important, but predictive markers of QoL have not been identified. We investigated the relationship between the serum testosterone (T) level before ENZ treatment and QoL changes in CRPC patients. PATIENTS AND METHODS: This prospective study was conducted between 2014 and 2018 at Gunma University Hospital and related facilities. We analyzed 95 patients in whom QoL could be evaluated using the Functional Assessment of Cancer Therapy-Prostate (FACT-P) questionnaire at baseline, and after 4 and 12 weeks of ENZ treatment. Serum T levels were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: The study population of 95 patients had a median age of 72 years and median prostate-specific antigen level of 21.6 ng/mL. The median overall survival from the commencement of ENZ treatment was 26.8 months. The median serum T level before ENZ treatment was 50.0 pg/mL. The mean total FACT-P scores at baseline, and after 4 and 12 weeks of ENZ treatment, were 95.8, 91.7, and 90.1, respectively. Differences in FACT-P scores between the high T level (High-T) group and low T level (Low-T) group (distinguished based on median split of the T level) were examined. The mean FACT-P scores were significantly higher in the High-T than Low-T group after both 4 and 12 weeks of ENZ treatment (98.5 vs. 84.6 and 96.4 vs. 82.2, respectively, both p < 0.05). The mean FACT-P score was significantly lower in the Low-T group after 12 weeks than before ENZ treatment (p < 0.05). CONCLUSION: The serum T level before treatment may be useful for predicting QoL changes after ENZ treatment in CRPC patients.

Two cases of CRPC with BRCA mutation treated by olaparib after favorable response to cisplatin.

Introduction: Several prostate cancers carry homologous recombination repair mutations that respond to olaparib. Because of the mechanism, the efficacy of platinum-based therapy can be used to predict the efficacy of poly(adenosine diphosphate-ribose) polymerase inhibitors such as olaparib. Case presentation: We experienced two neuroendocrine prostate cancer patients who achieved a response duration of more than 1 year with platinum-based therapy. Case 1 had a BRCA2 mutation in the germline and case 2 had a BRCA2 mutation in a somatic chromosome only. Both patients responded well to olaparib. Conclusion: Cisplatin and olaparib may overlap in response due to their medicinal action. It may be useful to consider genetic testing in some CRPC patients who have responded to cisplatin.

Changes in Renal Function of Patients With Prostate Cancer: Focus on Androgen Deprivation Therapy.

BACKGROUND/AIM: To investigate the association between androgen deprivation therapy (ADT) and changes in the estimated glomerular filtration rate (eGFR) in male Japanese patients with prostate cancer, based on post-hoc analysis of data from a previous prospective study. PATIENTS AND METHODS: Among 103 patients with prostate cancer in whom renal function changes were tracked over 5 years, 88 were divided into a group who completed ADT within 3 years (short ADT group; n=47) and a group who continued with ADT for more than 5 years (continuous ADT group; n=41). We compared the groups in terms of the eGFR, calculated based on age and serum creatinine (mg/dl) before ADT initiation and every other year over the next 5 years. RESULTS: The eGFR decreased by 4.91 and 2.89 ml/min in the short and continuous ADT groups, respectively, over the 5-year period following ADT initiation. The respective decreases in the eGFR were 0.98 and 0.58 ml/min/year. No significant difference in the eGFR was observed between the two groups at any measurement point. Patients treated with ADT showed a decrease in the eGFR of 0.58-0.98 ml/min/year over a 5-year period, which is about twice as high as that of normal Japanese males. No significant difference in the eGFR by ADT duration was observed. CONCLUSION: The eGFR decreased by 0.58-0.98 ml/min/year in our ADT patients, which was about twice as high as the rate of decrease in normal Japanese males, and approximately the same as in urine protein-positive male patients. We suggest that a large decrease in the eGFR may not play a role in the development of acute kidney injury. In addition, the duration of ADT does not appear to have a significant effect on the eGFR.

Fibroblast growth factor receptor inhibitor erdafitinib promotes Mcl-1 degradation and synergistically induces apoptosis with Bcl-xL/Bcl-2 inhibitor in urothelial cancer cells.

Metastatic urothelial cancer is a lethal disease. Although recent advances in immunotherapies and targeted therapy against fibroblast growth factor receptor (FGFR)2/3 mutation (erdafitinib) have improved patient survival, there is still a critical need for novel therapeutic strategies for patients who do not benefit from these treatments. Evasion of apoptosis through amplifying anti-apoptotic Bcl-2 family proteins (Mcl-1, Bcl-xL, Bcl-2) is one mechanism responsible for treatment resistance in urothelial cancers, suggesting that targeting anti-apoptotic proteins may be a possible therapeutic strategy for urothelial cancers. Here, we showed that erdafitinib increased Mcl-1 degradation mainly through previously unknown mechanisms and synergized with a BH3 mimetic drug targeting Bcl-xL/Bcl-2 to induce apoptosis in FGFR wild-type urothelial cancer cells. Strikingly, clinical sequencing data showed amplification of MCL1 or BCL2L1 (encoding Bcl-xL) in subsets of FGFR mutation-negative bladder cancer tissues. In conclusion, these findings suggest that exploiting apoptosis pathways may be a promising treatment strategy for patients with FGFR wild-type metastatic urothelial cancer with Mcl-1 or Bcl-xL overexpression.

Variants in HOXB13, G132E and F127C, Are Associated With Prostate Cancer Risk in Japanese Men.

BACKGROUND/AIM: Several studies have reported on the relationship between HOXB13 variants and an increased prostate cancer (PC) risk. To our knowledge there are not many studies on HOXB13 mutations in Japanese patients with prostate cancer, and there many issues remain uninvestigated. We herein clarified the association between HOXB13 genetic variants and PC risk in a Japanese population. PATIENTS AND METHODS: PC patients were diagnosed at the Gunma University Hospital and affiliated hospitals from 1994 to 2016. Sanger sequencing was performed on the coding regions of the HOXB13 gene in 152 familial PC (FPC) patients. Genotyping was performed on single nucleotide variants (SNVs) found in Sanger sequencing in 230 FPC patients from 152 pedigrees and 197 sporadic PC (SPC) patients and 144 controls. Allelic frequency and clinical data for each variant were studied in cases and controls. RESULTS: G132E and F127C were identified in FPC patients. The frequencies of G132E and F127C were significantly higher compared to the control group (p=0.039). In three families, seven PC patients shared the G132E variant, within second-to-third-degree relatives. It was not possible to clarify to pathogenicity of each SNV alone. CONCLUSION: We found two significant variants of the HOXB13 gene, G132E, F127C by analyzing and comparing gene samples from PC and non-PC patients. Furthermore, the HOXB13 G132E variant was found significantly increased in the FPC group.

Impact of freeze-thaw-induced pit aspiration on stem water transport in the subalpine conifer Abies veitchii.

During winter, subalpine conifers experience frequent freeze-thaw cycles in stem xylem that may cause embolism and pit aspiration due to increased water volume during the sap to ice transition. This study examined the occurrence and ecological impacts of a combination of freeze-thaw-induced pit aspiration and embolism triggered by natural and artificial stem freezing. In subalpine Veitch's fir (Abies veitchii) trees, the fraction of closed pits and embolized tracheids as well as conductivity losses were measured to examine pit aspiration and its effects. When trees incurred mild drought stress in February and early March, 70%-80% of stem conductivity was lost. Cryo-scanning electron microscopy indicated <20% embolized tracheids but ∼90% closed pits. Severe drought stress in late March caused 96% ± 1.2% (mean ± standard error) loss of stem conductivity, while the fraction of embolized tracheids increased to 64% ± 6.6%, and aspirated pit fraction decreased to 23% ± 5.6%. Experimental freeze-thaw cycles also increased pit aspiration from 7.1% ± 0.89% to 49% ± 10%, and the fraction of closed pits was positively correlated to the percent loss of stem hydraulic conductivity. The results indicated that freezing-induced pit aspiration is an important factor for stem xylem dysfunction under mild drought, and upon severe drought in winter; stem water transport is predominantly inhibited by xylem embolism.

Double-layer omics analysis of castration- and X-ray-resistant prostate cancer cells.

Castration-resistant prostate cancer shows resistance to not only androgen deprivation therapy (ADT) but also X-ray therapy. On the other hand, carbon ion beams have a high biological effect and are used for various cancers showing resistance to X-ray therapy. The purposes of this study are to clarify the difference in the sensitivity of Castration-resistant prostate cancer to X-ray and carbon ion beams and to elucidate the mechanism. The androgen-insensitive prostate cancer cell line LNCaP-LA established by culturing the androgen-sensitive prostate cancer cell line LNCaP for 2 years in androgen-free medium was used for this study. First, colony formation assays were performed to investigate its sensitivity to X-ray and carbon ion beams. Next, DNA mutation analysis on 409 cancer-related genes and comprehensive transcriptome analysis (RNA-seq) were performed with a next-generation sequencer. Lethal dose 50 values of X-rays for LNCaP and LNCaP-LA were 1.4 Gy and 2.8 Gy, respectively (P < 0.01). The Lethal dose 50 values of carbon ion beams were 0.9 Gy and 0.7 Gy, respectively (P = 0.09). On DNA mutation analysis, AR mutation was observed specifically in LNCaP-LA. From RNA-seq, 181 genes were identified as differentially expressed genes (DEGs; FDR <0.10, P < 0.00076) between LNCaP and LNCaP-LA. Function analysis suggested that cell death was suppressed in LNCaP-LA, and pathway analysis suggested that the NRF2-pathway involved in intracellular oxidative stress prevention was activated in LNCaP-LA. LNCaP-LA showed X-ray resistance compared to LNCaP and sensitivity to carbon ion beams. The AR mutation and the NRF2-pathway were suggested as causes of resistance.

Rapid stomatal closure contributes to higher water use efficiency in major C4 compared to C3 Poaceae crops.

Understanding water use characteristics of C3 and C4 crops is important for food security under climate change. Here, we aimed to clarify how stomatal dynamics and water use efficiency (WUE) differ in fluctuating environments in major C3 and C4 crops. Under high and low nitrogen conditions, we evaluated stomatal morphology and kinetics of stomatal conductance (gs) at leaf and whole-plant levels in controlled fluctuating light environments in four C3 and five C4 Poaceae species. We developed a dynamic photosynthesis model, which incorporates C3 and C4 photosynthesis models that consider stomatal dynamics, to evaluate the contribution of rapid stomatal opening and closing to photosynthesis and WUE. C4 crops showed more rapid stomatal opening and closure than C3 crops, which could be explained by smaller stomatal size and higher stomatal density in plants grown at high nitrogen conditions. Our model analysis indicated that accelerating the speed of stomatal closure in C3 crops to the level of C4 crops could enhance WUE up to 16% by reducing unnecessary water loss during low light periods, whereas accelerating stomatal opening only minimally enhanced photosynthesis. The present results suggest that accelerating the speed of stomatal closure in major C3 crops to the level of major C4 crops is a potential breeding target for the realization of water-saving agriculture.

Combination therapy with novel androgen receptor antagonists and statin for castration-resistant prostate cancer.

BACKGROUND: One of the growth mechanisms of castration-resistant prostate cancer (CRPC) is de novo androgen synthesis from intracellular cholesterol, and statins may be able to inhibit this mechanism. In addition, statins have been reported to suppress the expression of androgen receptors (ARs) in prostate cancer cell lines. In this study, we investigated a combination therapy of novel AR antagonists and statin, simvastatin, for CRPC. METHODS: LNCaP, 22Rv1, and PC-3 human prostate cancer cell lines were used. We developed androgen-independent LNCaP cells (LNCaP-LA). Microarray analysis was performed, followed by pathway analysis, and mRNA and protein expression was evaluated by quantitative real-time polymerase chain reaction and Western blot analysis, respectively. Cell viability was determined by MTS assay and cell counts. All evaluations were performed on cells treated with simvastatin and with or without AR antagonists (enzalutamide, apalutamide, and darolutamide). RESULTS: The combination of darolutamide and simvastatin most significantly suppressed proliferation in LNCaP-LA and 22Rv1 cells. In a 22Rv1-derived mouse xenograft model, the combination of darolutamide and simvastatin enhanced the inhibition of cell proliferation. In LNCaP-LA cells, the combination of darolutamide and simvastatin led to reduction in the mRNA expression of the androgen-stimulated genes, KLK2 and PSA; however, this reduction in expression did not occur in 22Rv1 cells. The microarray data and pathway analyses showed that the number of differentially expressed genes in the darolutamide and simvastatin-treated 22Rv1 cells was the highest in the pathway termed "role of cell cycle." Consequently, we focused our efforts on the cell cycle regulator polo-like kinase 1 (PLK1), cyclin-dependent kinase 2 (CDK2), and cell cycle division 25C (CDC25C). In 22Rv1 cells, the combination of darolutamide and simvastatin suppressed the mRNA and protein expression of these three genes. In addition, in PC-3 cells (which lack AR expression), the combination of simvastatin and darolutamide enhanced the suppression of cell proliferation and expression of these genes. CONCLUSIONS: Simvastatin alters the expression of many genes involved in the cell cycle in CRPC cells. Thus, the combination of novel AR antagonists (darolutamide) and simvastatin can potentially affect CRPC growth through both androgen-dependent and androgen-independent mechanisms.

Ratio of the expression levels of androgen receptor splice variant 7 to androgen receptor in castration refractory prostate cancer.

In clinical samples, the expression of androgen receptor (AR) and of AR splice variant 7 (AR-V7) is higher in castration-resistant prostate cancer (CRPC) compared with that in hormone-sensitive prostate cancer (PCa). However, there are only a few reports on the ratio of the expression levels of AR-V7 to AR (AR-V7/AR) in prostate tissue. The present study evaluated AR-V7/AR expression in various types of human prostate tissues and CRPC cells. Pretreatment prostate tissue samples from patients with benign prostatic hyperplasia (BPH; n=18), Gleason score 7 (n=17), and Gleason score 8-10 (n=26) were collected at the time of prostate biopsy, and tissue samples from CRPC patients (n=10) were collected at the time of transurethral resection of the prostate. Furthermore, androgen-independent LNCaP cells were established. The mRNA expression levels of AR and AR-V7, cell proliferation and prostate-specific antigen (PSA) production were evaluated by reverse transcription quantitative PCR, MTS assay and chemiluminescent enzyme immunoassay, respectively. There was a significant difference in AR-V7/AR expression ratios between the CRPC group and the BPH and pre-treatment PCa groups (CRPC, 7%; BPH and pre-treatment PCa, 1%). Subsequently, we compared the AR and AR-V7 expression levels in CRPC samples with those in the pretreatment prostate tissues from the same patients. The results demonstrated that the AR-V7/AR ratio increased from 3 to 9% after CRPC onset. Furthermore, in vitro experiment demonstrated that AR-V7 expression in LNCaP cells was increased after transforming into CRPC cells. The AR-V7/AR ratio also increased from 0.05 to 0.3%. In addition, small interfering (si)-RNA-mediated knockdown of AR inhibited the proliferation of and PSA production from androgen-independent LNCaP cells; however, AR-V7 knockdown had no effect. Conversely, siRNA-mediated knockdown of both AR and AR-V7 inhibited the proliferation of VCAP cells. In summary, the findings from the present study demonstrated that AR-V7 expression and AR-V7/AR ratio were increased after the onset of CRPC, which had a limited role in CRPC cell proliferation. Further investigation is required to clarify the roles of AR other splice variants and AR-V7 in CRPC.

Presepsin as a predictor of septic shock in patients with urinary tract infection.

BACKGROUND: Recently, presepsin has been reported to be a useful biomarker for early diagnosis of sepsis and evaluation of prognosis in septic patients. However, few reports have evaluated its usefulness in patients with urinary tract infections (UTI). This study aimed to evaluate whether presepsin could be a valuable marker for detecting severe sepsis, and whether it could predict the therapeutic course in patients with UTI compared with markers already used: procalcitonin (PCT) and C-reactive protein (CRP). METHODS: From April 2014 to December 2016, a total of 50 patients with urinary tract infections admitted to Gunma university hospital were enrolled in this study. Vital signs, presepsin, PCT, CRP, white blood cell (WBC) count, causative agents of urinary-tract infections, and other data were evaluated on the enrollment, third, and fifth days. The patients were divided into two groups: with (n = 11) or without (n = 39) septic shock on the enrollment day, and with (n = 7) or without (n = 43) sepsis on the fifth day, respectively. Presepsin was evaluated as a biomarker for systemic inflammatory response syndrome (SIRS) or septic shock. RESULTS: Regarding the enrollment day, there was no significant difference of presepsin between the SIRS and non-SIRS groups (p = 0.276). The median value of presepsin (pg/mL) was significantly higher in the septic shock group (p < 0.001). Multivariate logistic regression analysis showed that presepsin (≥ 500 pg/ml) was an independent risk factor for septic shock (p = 0.007). ROC curve for diagnosing septic shock indicated an area under the curve (AUC) of 0.881 for presepsin (vs. 0.690, 0.583, and 0.527 for PCT, CRP and WBC, respectively). Regarding the 5th day after admission, the median presepsin value on the enrollment day was significantly higher in the SIRS groups than in the non-SIRS groups (p = 0.006). On the other hand, PCT (≥ 2 ng/ml) on the enrollment day was an independent risk factor for SIRS. ROC curve for diagnosing sepsis on the fifth day indicated an AUC of 0.837 for PCT (vs. 0.817, 0.811, and 0.802 for presepsin, CRP, and WBC, respectively). CONCLUSIONS: This study showed that presepsin may be a good marker for diagnosing septic shock based on admission data in patients with UTI.