Masaki Nogawa

Antitumor Activity of Small Interfering RNA/Cationic Liposome Complex in Mouse Models of Cancer

Purpose: The RNA interference effect is an alternative to antisense DNA as an experimental method of down-regulating a specific target protein. Although the RNA interference effect, which is mediated by small interfering RNA (siRNA) or micro-RNA, has potential application to human therapy, the hydrodynamic method usually used for rapid administration of oligonucleotides is unsuitable for use in humans. In this study, we have investigated the antitumor activity of a synthetic siRNA, B717, which is sequence specific for the human bcl-2 oncogene, complexed with a novel cationic liposome, LIC-101. Experimental Design: In a mouse model of liver metastasis, we administered B717/LIC-101 by bolus intravenous injection, adjusting the rate and volume of administration to what is feasible in human therapy. In a mouse model bearing prostate cancer in which the cells were inoculated under the skin, B717/LIC-101 was administered subcutaneously around the tumor. Results: The B717/LIC-101 complex inhibited the expression of bcl-2 protein and the growth of tumor cell lines in vitro in a sequence-specific manner in the concentration range of 3 to 100 nmol/L. Furthermore, the complex had a strong antitumor activity when administered intravenously in the mouse model of liver metastasis. B717 (siRNA) was shown to be delivered to tumor cells in the mouse liver, but only when complexed with LIC-101. The complex also inhibited tumor cell growth in the mouse model bearing prostate cancer. Conclusions: By combining siRNA with our cationic liposome, we overcame the difficulty of administering siRNA to animals in ways that can be applied in human therapy. Although our siRNA/liposome complex is not yet in clinical trials, it is expected to provide a novel siRNA therapy for cancer patients.

Intravesical administration of small interfering RNA targeting PLK-1 successfully prevents the growth of bladder cancer

The mainstay in the management of invasive bladder cancer continues to be radical cystectomy. With regard to improvement of quality of life, however, therapies that preserve the bladder are desirable. We investigated the use of intravesical PLK-1 small interfering RNA (siRNA) against bladder cancer. Patients with bladder cancers expressing high levels of PLK-1 have a poor prognosis compared with patients with low expression. Using siRNA/cationic liposomes, the expression of endogenous PLK-1 could be suppressed in bladder cancer cells in a time- and dose-dependent manner. As a consequence, PLK-1 functions were disrupted. Inhibition of bipolar spindle formation, accumulation of cyclin B1, reduced cell proliferation, and induction of apoptosis were observed. In order to determine the efficacy of the siRNA/liposomes in vivo, we established an orthotopic mouse model using a LUC-labeled bladder cancer cell line, UM-UC-3(LUC). PLK-1 siRNA was successfully transfected into the cells, reduced PLK-1 expression, and prevented the growth of bladder cancer in this mouse model. This is the first demonstration, to our knowledge, of inhibition of cancer growth in the murine bladder by intravesical siRNA/cationic liposomes. We believe intravesical siRNA instillation against bladder cancer will be useful as a therapeutic tool.

Cytotoxic effects of γδ T cells expanded ex vivo by a third generation bisphosphonate for cancer immunotherapy

Nitrogen containing‐bisphosphonates (N‐BPs), widely used to treat bone diseases, have direct antitumor effects via the inactivation of Ras proteins. In addition to the direct antitumor activities, N‐BPs expand gdγδT cells, which exhibit major histocompatibility complex‐unrestricted lytic activity. BPs accumulate intermediate metabolites which may be tumor antigens in target cells. The purpose of our study was to clarify the cytotoxicity of gdγδ T cells expanded ex vivo by the most potent N‐BP, zoledronate (ZOL). Especially, we focused on the importance of pretreatment against target cells also with ZOL; 1 mμM ZOL plus IL‐2 increased the absolute number of gdγδT cells 298–768 fold for 14 days incubation. The small cell lung cancer and fibrosarcoma cell lines pretreated with 5 mμM ZOL showed a marked increase in sensitivity to lysis by gdγδT cells. While, untreated cell lines were much less sensitive to lysis by gdT cells. Video microscopy clearly demonstrated that gdγδT cells killed target cells pre‐treated with ZOL within 3 hr. Pretreatment with 80 mμg/kg ZOL also significantly enhanced the antitumor activity of gdγδT cells in mice xenografted with SBC‐5 cells. These findings show that ZOL significantly stimulated the proliferation of gdγδT cells and that gdγδT cells required pre‐treatment with ZOL for cytotoxic activity against target cells.

Antiproliferative efficacy of the third-generation bisphosphonate, zoledronic acid, combined with other anticancer drugs in leukemic cell lines

Bisphosphonates are widely used to treat bone diseases and appear to possess antitumor activity. Moreover, we recently found that a third-generation bisphosphonate, zoledronic acid (ZOL), synergistically interacts with imatinib in vitro and in vivo to induce antileukemic activity, and others have reported that ZOL interacts synergistically with paclitaxel. Thus, the efficacy of other antileukemic agents combined with ZOL should be evaluated experimentally. In this study, we investigated the effects of concurrent and sequential combinations of ZOL with several commonly used antileukemic agents, including imatinib, on the in vitro growth of leukemia cell lines. As a complement to our previous finding that ZOL synergistically augments the effects of imatinib, we report here that ZOL acts additively when administered concurrently with hydroxyurea (HU), cytarabine (Ara-C), or daunorubicin (DNR) in some leukemic cell lines. Furthermore, one day of ZOL pretreatment augmented the sensitivity of imatinib and Ara-C. Therefore, concurrent or sequential administration of ZOL with imatinib, HU, Ara-C, or DNR may increase the efficacy of leukemia treatment.

p53-independent anti-tumor effects of the nitrogen-containing bisphosphonate zoledronic acid.

Zoledronic acid (ZOL), a nitrogen‐containing bisphosphonate, exerts anti‐tumor effects by inhibiting the prenylation of small GT‐Pases. We have also reported that ZOL shows an anti‐leukemic effect by inducing apoptosis throughout the S phase to the G2/M boundary. Here, we studied the effects of ZOL on various cell cycle regulators, including p53, cyclin‐dependent kinases (CDKs), CDK inhibitors and cyclins, using BV173 leukemia and HCT116 colorectal carcinoma cell lines, harboring wild‐type (wt‐) p53. ZOL induced the accumulation of neither p53 nor p21WAF1/CIP1 during the execution of apoptosis in BV173 cells. Therefore, we investigated the dependence of ZOL‐induced apoptosis on intact p53 by using wt‐p53 HCT116 and a p53‐degraded HCT116 subline, and observed no significant difference. p57KIP2 was upregulated by ZOL in BV173 cells, but not in HCT116 cells. Flow cytometric analyses showed that ZOL also impaired the cell cycle‐dependent expression patterns of cyclins A, B and D3 in BV173. In conclusion, the p53‐independent anti‐tumor activities of ZOL suggest that it may be an attractive agent for treating cancers, including those with chemoresistance resulting from the loss of p53 function. ZOL also affected the coordinate expression patterns of several cell cycle regulators during the execution of anti‐tumor activity.

Rapid quantitation of immunoglobulin G antibodies specific for blood group antigens A and B by surface plasmon resonance

BACKGROUND:  The measurement of immunoglobulin (Ig) G blood group A/B antibody(anti‐A/B) levels is important for ABO‐unmatched organ recipients because the effective removal of the antibodies improves their prognosis. Currently existing methods to detect IgG anti‐A/B suffer limitations owing to high costs, low throughput, and poor adaptability to automation.

A novel tumor-related protein, c7orf24, identified by proteome differential display of bladder urothelial carcinoma

Proteome analysis of bladder cancer with narrow‐range pH 2‐DE has identified a novel protein on chromosome 7 encoded by ORF 24 (C7orf24) as one of the highly expressed proteins in cancer cells. C7orf24 is currently registered in the protein database as a hypothetical protein with unknown function. The homologs of C7orf24 in other animals have also been registered as putative protein genes. Western blot analysis using a mAb against C7orf24 confirmed its higher expression in bladder cancer compared with normal tissue. Several other cancer cell lines were also found to express C7orf24. However, the introduction of C7orf24 into Rat‐1 or NIH3T3 cells did not cause malignant transformation. A stable transfectant of NIH3T3 cells with recombinant retrovirus vector was produced for a growth rate assay, and a higher growth rate was observed in C7orf24‐expressing cells compared with the controls. Six kinds of small interfering RNAs (siRNAs) were then produced, and C7orf24‐siRNA#5 showed a strong knockdown effect on protein expression and significant antiproliferative effects on cancer cell lines were demonstrated by the MTT assay. Therefore, C7orf24 may have an important role in cancer cell proliferation, and may be an appropriate therapeutic target molecule against cancer.

Zoledronic acid mediates Ras-independent growth inhibition of prostate cancer cells.

Zoledronic acid (ZOL), the most potent known bisphosphonate, is clinically efficacious against advanced prostate cancer, although the molecular mechanism by which bisphosphonates prevent prostate cancer cell growth remains unknown. Because Ras is the most thoroughly characterized member of the small G-proteins involved in the regulation of many cellular functions including several oncogenic pathways, the aim of this study was to clarify whether Ras is the molecular target of ZOL in prostate cancer cells. The prostate cancer cell lines PC-3, DU145, and LNCaP were used. Cell proliferation was determined by a modified MTT assay. Geranylgeranyol (GGOH) and famesol (FOH) were used as analogues of geranylgeranyl-pyrophosphate and farnesyl-pyrophosphate, respectively. Changes in expression and/or membrane localization of Ras, Rap1, and phosphorylated MAPK were evaluated by Western blotting. ZOL mediated growth inhibition of prostate cancer cells in a dose- and time-dependent manner. The ZOL-induced growth inhibitory effect was circumvented by the addition of GGOH. In contrast, FOH did not reverse the growth inhibitory effect of ZOL. The amount of membrane-anchored Ras was clearly independent of ZOL-mediated growth inhibition. Unexpectedly, ZOL induced N- and H-Ras expression of the cytosolic fraction. Ras does not appear to be the molecular target for ZOL-induced growth inhibition. Prevention of geranylgeranylation rather than farnesylation is an important therapeutic target in prostate cancer.

A third-generation bisphosphonate, minodronic acid (YM529), successfully prevented the growth of bladder cancer in vitro and in vivo

Minodronic acid (YM529) is a third-generation bisphosphonate (BP) that has been shown to directly and indirectly prevent proliferation, induce apoptosis, and inhibit metastasis of various types of cancer cells. In this study, we have investigated the therapeutic efficacy of YM529 against bladder cancer, both in vitro and in vivo. YM529 inhibited geranylgeranylation as well as farnesylation and reduced the growth of all seven bladder cancer cell lines in a dose- and time-dependent manner in vitro. YM529 demonstrated a good synergistic or additive antiproliferative effect when administered in combination with cisplatin or paclitaxel. Immunohistochemical study revealed YM529 inhibited the prenylation of Rap1A in vivo. YM529 administered systemically did not markedly inhibit the growth of visceral metastases but it showed a significant anticancer effect on bone metastases monitored by an in vivo imaging system. Moreover, intravesical YM529 demonstrated significant growth inhibition in a bladder cancer orthotopic model. No adverse effects were associated with the systemic as well as the intravesical treatment regimens. In conclusion, our study suggests that YM529 may be a potent anticancer agent for bladder cancer. The efficacy and safety of this BP as an agent for combination chemotherapies against bladder cancer should be verified by early-phase clinical trials.

RAPID QUANTITATION OF IgG ANTIBODIES SPECIFIC FOR BLOOD GROUP ANTIGENS A AND B BY SURFACE PLASMON RESONANSE

The measurement of anti-blood group A/B (anti-A/B) IgG antibody levels is important for ABO unmatched-organ recipients because the effective removal of the antibodies improves their prognosis. However, currently existing methods to detect anti-A/B IgG antibodies suffer limitations due to high costs, low throughput, and poor adaptability to automation. We have developed a rapid means to quantitate anti-A/B IgG antibodies by surface plasmon resonance (SPR). The change in titers when the same plasma was diluted was precisely reflected by the SPR method. The coefficients of correlation between SPR and test tube (TT) methods for anti-A and -B IgG antibodies were 0.85 and 0.56, respectively. The SPR values also paralleled the TT values that showed a decline in titers after the removal of antibodies by double filtration plasma apheresis or plasma exchange.**This article is partly based on a study first reported in the Transfusion (Kimura, S., Yurugi, K., Yuasa, T., Tsuji, H., Segawa, H., Kuroda, J., Sato, K., Nogawa, M., Egawa, H., Tanaka, K., Maekawa, T.: Rapid quantitation of IgG antibodies specific for blood group antigens A and B by surface plasmon resonance. Transfusion, 45 (1): 56-62, 2005.)