Yoshikatsu Koga

MicroRNA Expression Profiling of Exfoliated Colonocytes Isolated from Feces for Colorectal Cancer Screening

To reduce the colorectal cancer (CRC) mortality rate, we have reported several CRC screening methods using colonocytes isolated from feces. Expression analysis of oncogenic microRNA (miRNA) in peripheral blood was recently reported for CRC detection. In the present study, we conducted miRNA expression analysis of exfoliated colonocytes isolated from feces for CRC screening. Two hundred six CRC patients and 134 healthy volunteers were enrolled in the study. miRNA expression of the miR-17-92 cluster, miR-21, and miR-135 in colonocytes isolated from feces as well as frozen tissues was analyzed by quantitative real-time PCR. The expression of the miR-17-92 cluster, miR-21, and miR-135 was significantly higher in CRC tissues compared with normal tissues. The exfoliated colonocytes of 197 CRC patients and 119 healthy volunteers were analyzed because of the presence of sufficient miRNA concentration. miR-21 expression did not differ significantly between CRC patients and healthy volunteers (P = 0.6). The expression of miR-17-92 cluster and miR-135 was significantly higher in CRC patients than in healthy volunteers (P < 0.0001). The overall sensitivity and specificity by using miRNA expression was 74.1% (146/197; 95% confidence interval, 67.4-80.1) and 79.0% (94/119; 95% confidence interval, 70.6-85.9), respectively. Sensitivity was dependent only on tumor location (P = 0.0001). miRNA was relatively well conserved in exfoliated colonocytes from feces both of CRC patients and healthy volunteers. miRNA expression analysis of the isolated colonocytes may be a useful method for CRC screening. Furthermore, oncogenic miRNA highly expressed in CRC should be investigated for CRC screening tests in the future. Cancer Prev Res; 3(11); 1435–42. ©2010 AACR.

Potent antitumor effect of SN‐38‐incorporating polymeric micelle, NK012, against malignant glioma

Recent published reports on clinical trials of CPT‐11 indicate the effectiveness of this compound, a prodrug of SN‐38, against malignant glioma in combination with anti‐vascular endothelial growth factor antibody. Here, we determined if NK012, and SN‐38 incorporating micelle, can be an appropriate formulation for glioblastoma treatment compared with CPT‐11. In vitro cytotoxicity was evaluated against several glioma lines with NK012, CPT‐11, SN‐38, ACNU, CDDP and etoposide. For the in vivo test, a human glioma line (U87MG) transfected with the luciferase gene was inoculated into nude mice brain for pharmacokinetic analysis by fluorescence microscopy and high‐performance liquid chromatography after intravenous injection of NK012 and CPT‐11. In vivo antitumor activity of NK012 and CPT‐11 was evaluated by bioluminescence image and Kaplan‐Meier analyses. The growth‐inhibitory effects of NK012 were 34‐ to 444‐fold more potent than those of CPT‐11. Markedly enhanced and prolonged distribution of free SN‐38 in the xenografts was observed after NK012 injection compared with CPT‐11. NK012 showed significantly potent antitumor activity against an orthotopic glioblastoma multiforme xenograft and significantly longer survival rate than CPT‐11 (p = 0.0014). This implies that NK012 can pass through the blood brain tumor barrier effectively. NK012, which combines enhanced distribution with prolonged sustained release, may be ideal for glioma treatment. Currently, a phase I study of NK012 is almost complete in Japan and the US. The present translational study warrants the clinical phase II study of NK012 in patients with malignant glioma.

Exosome can prevent RNase from degrading microRNA in feces

BACKGROUND Because the stability of miRNA in feces has not been clarified, we examined the stability of miRNA in feces. METHODS RNase was added into culture media of HT-29 cells and fecal homogenates. The relative quantifications of miRNA were analyzed by real-time RT-PCR. RESULTS Cellular miRNA or exosomal miRNA were protected from RNase by the cellular membrane or the exosome; meanwhile, free miRNA was degraded immediately and completely by RNase. CONCLUSION The present study revealed that exosome or cellular membrane could prevent RNase from degrading miRNA inside the exosome or cells even in a dreadful condition, as in feces.

NC-6300, an epirubicin-incorporating micelle, extends the antitumor effect and reduces the cardiotoxicity of epirubicin

Epirubicin is widely used to treat various human tumors. However, it is difficult to achieve a sufficient antitumor effect because of dosage limitation to prevent cardiotoxicity. We hypothesized that epirubicin‐incorporating micelle would reduce cardiotoxicity and improve the antitumor effect. NC‐6300 comprises epirubicin covalently bound to PEG polyaspartate block copolymer through an acid–labile hydrazone bond. The conjugate forms a micellar structure of 40–80 nm in diameter in an aqueous milieu. NC‐6300 (10, 15 mg/kg) and epirubicin (10 mg/kg) were given i.v. three times to mice bearing s.c. or liver xenograft of human hepatocellular carcinoma Hep3B cells. Cardiotoxicity was evaluated by echocardiography in C57BL/6 mice that were given NC‐6300 (10 mg/kg) or epirubicin (10 mg/kg) in nine doses over 12 weeks. NC‐6300 showed a significantly potent antitumor effect against Hep3B s.c. tumors compared with epirubicin. Moreover, NC‐6300 also produced a significantly longer survival rate than epirubicin against the liver orthotopic tumor of Hep3B. With respect to cardiotoxicity, epirubicin‐treated mice showed significant deteriorations in fractional shortening and ejection fraction. In contrast, cardiac functions of NC‐6300 treated mice were no less well maintained than in control mice. This study warrants a clinical evaluation of NC‐6300 in patients with hepatocellular carcinoma or other cancers.

Enhanced distribution of NK012, a polymeric micelle-encapsulated SN-38, and sustained release of SN-38 within tumors can beat a hypovascular tumor

Human pancreatic cancer is generally hypovascular in nature and rich in interstitium. These pathological barriers may contribute to the intractable nature of pancreatic cancer by binding the penetration of anticancer agents throughout the tumor tissue. The aim of the present study was to determine whether NK012 is an appropriate formulation for the treatment of hypovascular tumors. Among pancreatic tumor xenografts, PSN1 appeared to have the richest tumor vasculature and the least number of stromal cells and matrix. In contrast, Capan1 had the poorest tumor vasculature and most abundant stromal tissue. Fluorescence microscopy and high‐performance liquid chromatography analysis demonstrated that although NK012 accumulated and continued to be distributed for more than 48 h throughout the entire body of both tumors, CPT‐11 disappeared almost entirely from both tumors within 6 h. In addition, efficient sustained release of SN‐38 was maintained for more than 96 h in both tumors following administration of NK012. Following the administration of CPT‐11, SN‐38 was no longer detectable after 24 h in the Capan1 tumor or after 48 h in the PSN1 tumor. All tumors were eradicated in the mice treated with NK012 but not in those treated with CPT‐11. Because the antitumor activity of SN‐38 is time dependent, NK012, which combines enhanced distribution with sustained release of SN‐38 within tumors, may be ideal for the treatment of hypovascular tumors, such as pancreatic cancer. (Cancer Sci 2008; 99: 1258–1264)

Fecal miR-106a Is a Useful Marker for Colorectal Cancer Patients with False-Negative Results in Immunochemical Fecal Occult Blood Test

Background: Immunochemical fecal occult blood test (iFOBT) is widely used for colorectal cancer screening; however, its sensitivity is insufficient. We recently reported a fecal microRNA (miRNA) test (FmiRT) to detect colorectal cancer. In this study, we investigated a new colorectal cancer screening method combining iFOBT and FmiRT to improve the sensitivity compared with iFOBT alone. Methods: In total, 117 colorectal cancer patients and 107 healthy volunteers were enrolled. Ten-milligram fecal samples were collected and iFOBT was conducted. Fecal RNA was extracted from residuum of iFOBT and then the expression of 14 kinds of miRNA was analyzed for the FmiRT using real-time reverse transcription PCR. Results: Levels of fecal miR-106a expression in iFOBT+ patients and iFOBT− patients were significantly higher than in healthy volunteers (P = 0.001). The sensitivity and specificity of FmiRT using miR-106a were 34.2% and 97.2%, and those of iFOBT were 60.7% and 98.1%, respectively. The overall sensitivity and specificity of the new screening method combining iFOBT and FmiRT were 70.9% and 96.3%, respectively. One quarter of colorectal cancer patients with false-negative iFOBT seemed to be true positive upon adding FmiRT using fecal miR-106a. Conclusions: Fecal miR-106a is a good molecular marker to identify colorectal cancer patients from among those with negative iFOBT results. FmiRT combined with iFOBT may improve the sensitivity to detect colorectal cancer. Impact: We have shown the usefulness of fecal miR-106a to detect the colorectal cancer patients among those with negative iFOBT results. Cancer Epidemiol Biomarkers Prev; 22(10); 1844–52. ©2013 AACR.

Antitumor Activity of NK012 Combined with Cisplatin against Small Cell Lung Cancer and Intestinal Mucosal Changes in Tumor-Bearing Mouse after Treatment

Purpose: To investigate the advantages of treatment with the SN-38–incorporating polymeric micelles NK012 over CPT-11 in combination with cisplatin [cis-dichlorodiammineplatinum (II) (CDDP)] in mice bearing a small cell lung cancer xenograft in terms of antitumor activity and toxicity, particularly intestinal toxicity. Experimental Design: Cytotoxic effects were evaluated in human small cell lung cancer cell lines [H69, H82, and vascular endothelial growth factor (VEGF)–secreting cells (SBC-3/VEGF and its mock transfectant SBC-3/Neo)]. In vivo antitumor effects were evaluated in SBC-3/Neo–bearing and SBC-3/VEGF–bearing mice after NK012/CDDP or CPT-11/CDDP administration on days 0, 7, and 14. Drug distribution was analyzed by high-performance liquid chromatography or fluorescence microscopy, and the small intestine was pathologically examined. Results: The in vitro growth-inhibitory effects of NK012 were 198- to 532-fold more potent than those of CPT-11. A significant difference in the relative tumor volume on day 30 was found between NK012/CDDP and CPT-11/CDDP treatments (P = 0.0058). Inflammatory changes in the small intestinal mucosa were rare in all NK012-treated mice but were commonly observed in CPT-11–treated mice. Moreover, a large amount of CPT-11 was excreted into the feces and high CPT-11 concentration was detected in the small intestinal epithelium. On the other hand, a small amount of NK012 was found in the feces and NK012 was weakly and uniformly distributed in the mucosal interstitium. Conclusions: NK012/CDDP combination may be a promising candidate regimen against lung cancer without severe diarrhea toxicity and therefore warrants further clinical evaluation.

The significance of microscopic mass spectrometry with high resolution in the visualisation of drug distribution

The visualisation and quantitative analysis of the native drug distribution in a pre-clinical or clinical setting are desirable for evaluating drug effects and optimising drug design. Here, using matrix-assisted laser desorption ionisation imaging mass spectrometry (MALDI-IMS) with enhanced resolution and sensitivity, we compared the distribution of a paclitaxel (PTX)-incorporating micelle (NK105) with that of PTX alone after injection into tumour-bearing mice. We demonstrated optically and quantitatively that NK105 delivered more PTX to the tumour, including the centre of the tumour, while delivering less PTX to normal neural tissue, compared with injection with PTX alone. NK105 treatment yielded a greater antitumour effect and less neural toxicity in mice than did PTX treatment. The use of high-resolution MALDI-IMS may be an innovative approach for pharmacological evaluation and drug design support.

The inhibition of pancreatic cancer invasion-metastasis cascade in both cellular signal and blood coagulation cascade of tissue factor by its neutralisation antibody

Tissue factor (TF), the initiating cell surface receptor for the blood coagulation cascade, plays an important role in malignant transformation of the pancreas, although the precise mechanism remains unresolved. Here, we report that the TF - factor VIIa complex in human pancreatic cancer cells produced a significant amount of MMP-9 and promoted invasion ability in vitro and invasion and metastasis in vivo. For treatment, we successfully developed an anti-human TF monoclonal antibody that inhibits both cellular signalling and blood coagulation cascade via TF. Invasive capability and MMP-9 expression were significantly reduced by the antibody. The antibody inhibited not only tumour invasion in the orthotopic model, but also haematogenous metastasis in the portal-injection liver metastasis model. In conclusion, the TF-VIIa complex plays an important role in invasion-metastasis by enhancing tumour cell infiltration ability and forming microthrombi. The newly established anti-human TF neutralisation antibody may be useful for the treatment of pancreatic and other invasive cancers.

Effect of combined treatment with the epirubicin-incorporating micelles (NC-6300) and 1,2-diaminocyclohexane platinum (II)-incorporating micelles (NC-4016) on a human gastric cancer model.

Anticancer agent‐incorporating polymeric micelles accumulate effectively in tumors via the enhanced permeability and retention effect to exert potent antitumor effects. However, combined use of such micelles has not been elucidated. We compared the effect of combining the epirubicin‐incorporating micelle NC‐6300 and 1,2‐diaminocyclohexane platinum (II) (oxaliplatin parent complex)‐incorporating micelle NC‐4016 (NCs) with that of epirubicin and oxaliplatin (E/O) in 44As3Luc cells using the combination index method. The in vivo antitumor activities of NCs and E/O were evaluated in mice bearing 44As3Luc xenografts. Pharmacokinetic analysis was performed by high‐performance liquid chromatography and mass spectrometry. Cardiotoxicity of NC‐6300 and epirubicin was assessed by echocardiography. Neurotoxicity of NC‐4016 and oxaliplatin was evaluated by examining the paw withdrawal response to noxious mechanical stimuli. NCs showed a highly synergistic activity equivalent to E/O. In vivo, NCs exhibited higher antitumor activity in the subcutaneous tumor model and longer overall survival in the orthotopic tumor model than E/O (p < 0.001, p = 0.015, respectively). The intratumor concentrations of epirubicin and platinum were significantly higher following NCs than following E/O administration. Moreover, the micelles showed lower cardiotoxicity and neurotoxicity than the corresponding conventional drugs. The combined use of the micelles was associated with remarkable efficacy and favorable toxicities in the human gastric cancer model, and warrants the conduct of clinical trials.