Hidekazu Segawa

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.

Inhibition of leukemic cell growth by a novel anti-cancer drug (GUT-70) from calophyllum brasiliense that acts by induction of apoptosis

During our search for cancer chemopreventing compounds derived from plant sources, we discovered that the natural product GUT‐70, isolated from the stem bark of Calophyllum brasiliense collected in Brazil, significantly inhibits the growth of leukemic cells. GUT‐70, characterized as a tricyclic coumarin, 5‐methoxy‐2,2‐dimethyl‐6‐(2‐methyl‐1‐oxo‐2‐butenyl) ‐10‐propyl‐2H,8H‐benzo[1,2‐b;3,4‐b′]dipyran‐8‐one (C23H26O5), inhibited all 6 human leukemic cell lines evaluated, including the P‐glycoprotein overexpressing cell line, in a concentration and time‐dependent manner with IC50 values from 2–5 μM. Furthermore, GUT‐70 did not inhibit colony formation by normal hematopoietic progenitors up to 30 μM and also did not inhibit the proliferation of normal human hepatocytes up to 30 μM. GUT‐70 activated the caspase 2, 3, 8 and 9, and induced the apoptosis in leukemic cells, which was inhibited by caspase inhibitors. GUT‐70 induced anti‐leukemic effects independent of the p53‐p2lWAFl/CIP1 pathway and increased the overall expression of p27KIP1 and p57KIP2, to stop the cell cycle at the G1/S transition. Thus, a novel anti‐cancer drug, GUT‐70 isolated from the stem bark of C. brasiliense induces caspase‐mediated and p53‐independent apoptosis to overcome multidrug resistance and may become a potent leukemia therapeutics.

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.

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.

Zoledronate synergises with imatinib mesylate to inhibit Ph primary leukaemic cell growth.

We examined the in vivo effects and safety of the third generation bisphosphonate, zoledronate (ZOL) alone and combined with imatinib mesylate against primary Philadelphia chromosome positive (Ph+) leukaemic cells. ZOL inhibited the prenylation of Rap1A in leukaemic cells in vitro and synergised with imatinib to enhance the survival of mice engrafted with cells from imatinib‐responders, but not from non‐responders because of mutated BCR‐ABL. These findings suggest that the combination of ZOL and imatinib accelerate the eradication of Ph+ clone, resulting in better prognosis of Ph+ leukaemia patients who have not yet acquired mutations.

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.

MALT Lymphoma at the Base of the Tongue Developing without any Background of Immunodeficiency or Autoimmune Disease

We report a very rare case of a mucosa-associated lymphoid tissue (MALT) lymphoma of the base of the tongue. A 61-year-old woman was admitted to our hospital for further examination of a 12 mm × 15 mm × 5 mm tongue tumor. Histological examination of the tumor revealed a marked lymphoepithelial lesion. Lymphoma cells expressed CD5 (m ), CD10 (m ), CD19(+), CD20(+) on the surface of the cells by fluorescence activated cell sorter, and the genotypic analysis of the tumor cells revealed the presence of immunoglobulin heavy chain rearrangement and the absence of BCL-2 gene rearrangement by southern blot hybridization. Furthermore, neither the t(11;18) (q21;q21) translocation nor trisomy 3 was detected in lymphoma cells by fluorescence in situ hybridization method. The tongue tumor was completely resected and no recurrence has been noted in the 13 months to date.