Perforin secreted from cytotoxic lymphocytes plays a critical role in cancer immunosurveillance. The aim of this study was to investigate the therapeutic potential of liposomes containing perforin ...expression vector driven by the promotor of prostate-specific antigen (PSA). The anti-tumor effect of perforin was analyzed using prostate cancer (PC) PC-3 cells in which perforin expression was controlled by Tet-on system (PC-3PRF cells). Liposomes encapsulating PSA promoter-driven perforin expression vector (pLipo) were constructed for its specific expression in PC. The anti-tumor effect of pLipo was evaluated in vitro using docetaxel-resistant PC 22Rv1 PC cell line, 22Rv1DR, and PC-3 cells in the presence of human peripheral blood mono nuclear cells (PBMCs) and also in vivo using male nude mice bearing 22Rv1DR cell-derived tumor xenograft. Induction of perforin significantly inhibited growth of PC-3PRF cells. Treatment with pLipo induced perforin expression in 22Rv1DR cells expressing PSA but not in PC-3 cells lacking it. Treatment with pLipo at a low concentration was prone to inhibit growth of both cell lines and significantly inhibited growth of 22Rv1DR cells when co-incubated with PBMCs. The combined use of pLipo at a high concentration with PBMCs showed nearly complete inhibition of 22Rv1DR cell growth. Intravenous administration of pLipo via tail vein increased the level of perforin in tumor and serum and significantly decreased the tumor volume. Our results suggest that liposome-mediated PC-specific expression of perforin could be a novel therapy for advanced PC.
Abstract
We aimed to develop a sandwich ELISA to detect prostate-specific membrane antigen (PSMA) on small extracellular vesicles (EVs) using T-cell immunoglobulin domain and mucin domain-containing ...protein 4 (Tim4) as a capture molecule for EVs and to evaluate its diagnostic potential in urologic malignancies. First, we optimized the conditions for sandwich ELISA measuring the PSMA level on EVs captured from serum by Tim4 and found that the use of highly-purified EVs released from Tim4 that had captured EVs in serum reduced the background. Second, we confirmed its validity by studying mouse xenograft model for prostate cancer (PC). Lastly, we measured PSMA-EVs in serum of patients with urologic malignancies. The PSMA-EV levels were significantly higher in metastatic PC and castration-resistant PC (CRPC) patients than in therapy-naïve PC patients. In renal cell carcinoma (RCC) patients, PSMA-EVs were elevated in those with metastasis compared with those without metastasis, which may reflect the development of the neovasculature positive for PSMA in tumors. In conclusion, we developed a sandwich ELISA for detection of PSMA-EVs using highly-purified EVs isolated from serum by Tim4. Our results suggest that PSMA-EVs may be useful to diagnose and monitor not only PC but also RCC and possibly other hypervascular solid tumors.
Exosomes or extracellular vesicles have the potential as a diagnostic marker for various diseases including cancer. In order to identify novel exosomal markers for prostate cancer (PC), we performed ...proteomic analysis of exosomes isolated from PC cell lines and examined the usefulness of the marker in patients.
Exosomes isolated by differential centrifugation from the culture medium of androgen-dependent LNCaP prostate cancer cell line and its sublines of partially androgen-independent C4, androgen-independent C4-2 and bone metastatic C4-2B were subjected to iTRAQ-based proteomic analysis. Exosomes were also isolated by immunocapture and separated by size exclusion chromatography and density gradient centrifugation. Protein expression was determined by Western blot analysis. GGT activity was measured using a fluorescent probe, γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG). Immunohistochemical analysis of tissues was performed using anti-GGT1 antibody.
Among proteins upregulated in C4-2 and C4-2B cells than in LNCaP cells, we focused on gamma-glutamyltransferase 1 (GGT1), a cell-surface enzyme that regulates the catabolism of extracellular glutathione. The levels of both GGT1 large and small subunits were elevated in exosomes isolated from C4-2 and C4-2B cells by differential centrifugation and by immunocapture with anti-CD9 or -prostate-specific membrane antigen (PSMA) antibody. In cell lysates and exosomes, GGT1 expression correlated with GGT activity. Size exclusion chromatography of human serum demonstrated the presence of GGT activity and GGT1 subunits in fractions positive for CD9. Density gradient centrifugation revealed the co-presence of GGT1 subunits with CD9 in exosomes isolated by differential centrifugation from human serum. Since GGT activity correlated with GGT1 expression in serum exosomes isolated by differential centrifugation, we measured serum exosomal GGT activity in patients. Unexpectedly, we found that serum exosomal GGT activity was significantly higher in PC patients than in benign prostatic hyperplasia (BPH) patients. In support of this finding, immunohistochemical analysis showed increased GGT1 expression in PC tissues compared with BPH tissues.
Our results suggest that serum exosomal GGT activity could be a useful biomarker for PC.
COVID-19 causes physical and psychological impacts on health care workers (HCWs), especially when it occurs during an outbreak. As there are few reports on outcomes of HCWs infected with COVID-19 ...during a hospital outbreak, we investigated the physical and psychological impacts on HCWs infected with COVID-19 during an outbreak in our hospital.
During the outbreak in our hospital, 231 people were infected with COVID-19 including patients, HCWs and their families. Among them, 83 HCWs were enrolled in this study. Current quality of life (QOL) was assessed with the EuroQol-visual analogue scales (EQ-VAS), and motivation to keep on working was evaluated by a 10-point analogue scale. Physiological recovery rates including return to work (RTW) period were also analyzed.
One nurse quit work due to anxiety regarding re-infection with COVID-19. The median period to RTW from the diagnosis was 14.0 (12.0–17.0) days. Motivation to keep on working was slightly reduced, and the EQ-VAS was 75.0 (65.0–83.6). There were no significant differences in QOL and motivation between male and female HCWs, nurses and other HCWs, treatment and non-treatment group, and supplemental and non-supplemental oxygen group. The most frequent persistent symptoms at 1,3 and 6 months after infection were anosmia followed by fatigue.
Although QOL and motivation to keep on working were slightly reduced, only one HCW quit work. No severe persistent symptoms were observed, and the RTW period was relatively short.
Unlike urinary tract infection (UTI), asymptomatic bacteriuria (ABU) should not be treated, with some exceptions such as pregnant women and patients who will undergo traumatic urologic interventions. ...However, there has been no clinically available marker for their differential diagnosis. Exosomes or small extracellular vesicles carry proteins contained in cells from which they are derived, thus having the potential as a biomarker of several diseases. On the basis of the hypothesis that the molecular signature of exosomes in urine may differ between UTI and ABU patients, we examined if urinary exosomes could serve as a marker for their differential diagnosis. Exosomes were isolated by ultracentrifugation or affinity‐based method from cell culture medium of monocytic THP‐1 and uroepithelial SV‐HUC‐1 cells and human urine. Protein expression was examined by Western blot analysis, ELISA, and CLEIA. The results showed that the levels of intracellular signalling molecules Akt and ERK and transcription factor NF‐κB increased in exosomes isolated from THP‐1 and SV‐HUC‐1 cells cocultured with Escherichia coli and/or treated with lipopolysaccharide. In urinary exosomes of UTI patients, Akt significantly diminished, and an exosomal marker CD9 showed a trend to decrease after treatment with antimicrobial agents. More importantly, Akt and CD9 levels in urinary exosomes were higher in UTI patients than in ABU patients, which was also observed after correction by urine creatinine. Collectively, these results suggest that Akt and CD9 in urinary exosomes could be useful markers for differential diagnosis of UTI and ABU.
CC chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein-1) has been demonstrated to recruit monocytes to tumor sites. Monocytes are capable of being differentiated into ...tumor-associated macrophages (TAMs) and osteoclasts (OCs). TAMs have been shown to promote tumor growth in several cancer types. Osteoclasts have also been known to play an important role in cancer bone metastasis. To investigate the effects of CCL2 on tumorigenesis and its potential effects on bone metastasis of human prostate cancer, CCL2 was overexpressed into a luciferase-tagged human prostate cancer cell line PC-3. In vitro, the conditioned medium of CCL2 overexpressing PC-3luc cells (PC-3lucCCL2) was a potent chemoattractant for mouse monocytes in comparison to a conditioned medium from PC-3lucMock. In addition, CCL2 overexpression increased the growth of transplanted xenografts and increased the accumulation of macrophages in vivo. In a tumor dissemination model, PC-3lucCCL2 enhanced the growth of bone metastasis, which was associated with more functional OCs. Neutralizing antibodies targeting both human and mouse CCL2 inhibited the growth of PC-3luc, which was accompanied by a decrease in macrophage recruitment to the tumor. These findings suggest that CCL2 increases tumor growth and bone metastasis through recruitment of macrophages and OCs to the tumor site.
Docetaxel is first-line chemotherapy for castration-resistant prostate cancer (CRPC), but most patients acquire docetaxel resistance. CD44 has been shown to be involved in drug resistance of cancers ...including prostate cancer. We hypothesized that CD44 in serum exosomes could be a diagnostic marker for docetaxel resistance in CRPC patients. In this study, we examined CD44 protein and mRNA expression in cell lysates and exosomes isolated from prostate cancer cells, evaluated the effect of CD44v8-10 knockdown on docetaxel sensitivity and measured CD44 mRNA copy numbers contained in serum exosomes in prostate cancer patients.
Docetaxel-sensitive PC-3 prostate cancer cells and docetaxel-resistant PC-3R cells established previously from parental PC-3 cells were used. CD44v8-10 knockdown was performed by siRNA transfection. Blood was collected from 50 docetaxel-naïve and 10 docetaxel-resistant patients and 15 control males. CD44 protein expression was evaluated by Western blotting. CD44 mRNA expression was measured by RT-digital PCR.
The levels of CD44v8-10 protein and mRNA in cell lysates and exosomes were higher in PC-3R cells than in PC-3 cells. CD44v8-10 knockdown significantly increased docetaxel sensitivity of PC-3R cells. The CD44v8-10 mRNA copy numbers in serum exosomes were higher in docetaxel-resistant patients than in docetaxel-naïve patients and control males (median 46, 12 and 17 copies/mL serum, respectively, P = 0.032). In contrast, the serum exosomal mRNA copy numbers of CD44 standard isoform (CD44s) were not different among 3 groups (median 25, 14 and 13 copies/mL serum, respectively, P = 0.150).
CD44v8-10 may be involved in docetaxel resistance in prostate cancer and serum exosomal CD44v8-10 mRNA could be a diagnostic marker for docetaxel-resistant CRPC.
Cell biology; Molecular biology; Cancer research; Chemotherapy; Urology; Castration-resistant prostate cancer, CD44, Docetaxel resistance, Exosomes, Diagnostic marker
Radical cystectomy remains the gold standard for treatment of muscle‐invasive bladder cancer. Robot‐assisted radical cystectomy has technical advantages over laparoscopic radical cystectomy and has ...emerged as an alternative to open radical cystectomy. Despite the advancements in robotic surgery, experience with total intracorporeal reconstruction of urinary diversion remains limited. Most surgeons have carried out the hybrid approach of robot‐assisted radical cystectomy and extracorporeal reconstruction of urinary diversion, as intracorporeal reconstruction of urinary diversion remains technically challenging. However, intracorporeal reconstruction of urinary diversion might potentially proffer additional benefits, such as decreased fluid loss, reduction in estimated blood loss and a quicker return of bowel function. The adoption of intracorporeal ileal neobladder reconstruction has hitherto been limited to high‐volume academic institutions. In the present review, we compare the totally intracorporeal robot‐assisted radical cystectomy approach with open radical cystectomy and robot‐assisted radical cystectomy + extracorporeal reconstruction of urinary diversion in muscle‐invasive bladder cancer patients.
Cabazitaxel (CBZ) is now widely used for prostate cancer (PC) patients resistant to docetaxel (DOC), however, most patients eventually acquire resistance. It will, therefore, be of great benefit to ...discover novel therapeutic target for the resistance. We aimed to identify candidate therapeutic targets for CBZ-resistance by proteomic analysis of extracellular vesicles (EVs) isolated from serum of DOC-resistant PC patients who later developed CBZ-resistance as well as those harvested from culture medium of DOC- and CBZ-resistant PC cell lines.
Using T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4) conjugated to magnetic beads, EVs were purified from serum of PC patients with DOC-resistance that was collected before and after acquiring CBZ-resistance and conditioned medium of DOC-resistant (22Rv1DR) and CBZ-resistant (22Rv1CR) PC cell lines. Protein analysis of EVs was performed by nanoLC-MS/MS, followed by a comparative analysis of protein expression and network analysis. The cytotoxic effect of a phosphatidylinositol-3-kinase (PI3K) inhibitor, ZSTK474, was evaluated by WST-1 assay. The expression and phosphorylation of PI3K and PTEN were examined by western blot analysis.
Among differentially regulated proteins, 77 and 61 proteins were significantly increased in EVs from CBZ-resistant PC cell line and patients, respectively. A comparison between the two datasets revealed that six proteins, fructose-bisphosphate aldolase, cytosolic nonspecific dipeptidase, CD63, CD151, myosin light chain 9, and peroxiredoxin-6 were elevated in EVs from both cell line and patients. Network analysis of the increased EV proteins identified pathways associated with CBZ-resistance including PI3K signaling pathway. ZSTK474 significantly inhibited growth of 22Rv1CR cells and improved their sensitivity to CBZ. In 22Rv1CR cells, PI3K was activated and PTEN that inhibits PI3K was deactivated.
Proteomic analysis of serum EVs was successfully accomplished by using Tim-4 as a tool to isolate highly purified EVs. Our results suggest that the combination use of CBZ and PI3K inhibitor could be a promising treatment option for CBZ-resistant PC patients.
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