Yuri Kamitsuji

ABT-737 is a useful component of combinatory chemotherapies for chronic myeloid leukaemias with diverse drug-resistance mechanisms

The effect of ABT‐737, a BH3‐mimicking inhibitor for anti‐apoptotic Bcl‐2 and Bcl‐XL, but not Mcl‐1, against Bcr‐Abl‐positive (Bcr‐Abl+) leukaemic cells was examined. ABT‐737 potently induced apoptosis in Bcr‐Abl+ chronic myeloid leukaemia (CML) cell lines and primary CML samples in vitro and prolonged the survival of mice xenografted with BV173 cells, a CML cell line. Higher expression of anti‐apoptotic Bcl‐2 proteins reduced cell killing by ABT‐737 in each cell line, but there was no correlation between the sensitivities to ABT‐737 and the specific expression patterns of Bcl‐2 family proteins among cell lines. Thus, the cell killing effect of ABT‐737 must be determined not only by the expression patterns of Bcl‐2 family proteins but also by other mechanisms, such as high expression of Bcr‐Abl, or a drug‐efflux pump, in CML cells. ABT‐737 augmented the cell killing effect of imatinib in Bcr‐Abl+ cells with diverse drug‐resistance mechanisms unless leukaemic cells harboured imatinib‐insensitive Abl kinase domain mutations, such as T315I. The combination of homoharringtonine that reduces Mcl‐1 enhanced the killing by ABT‐737 strongly in Bcr‐Abl+ cells even with T315I mutation. These results suggest that ABT‐737 is a useful component of chemotherapies for CML with diverse drug‐resistance mechanisms.

Apoptosis-based dual molecular targeting by INNO-406, a second-generation Bcr-Abl inhibitor, and ABT-737, an inhibitor of antiapoptotic Bcl-2 proteins, against Bcr-Abl-positive leukemia.

Bcr-Abl is the cause of Philadelphia-positive (Ph+) leukemias and also constitutes their principal therapeutic target, as exemplified by dramatic effects of imatinib mesylate. However, mono-targeting of Bcr-Abl does not always achieve complete leukemia eradication, and additional strategies those enable complete elimination of leukemic cells are desired to develop. Here we demonstrate that INNO-406, a much more active Bcr-Abl tyrosine kinase inhibitor than imatinib, augments the activities of several proapoptotic Bcl-2 homology (BH)3-only proteins (Bim, Bad, Bmf and Bik) and induces apoptosis in Ph+ leukemia cells via Bcl-2 family-regulated intrinsic apoptosis pathway. ABT-737, an inhibitor of antiapoptotic Bcl-2 and Bcl-XL, greatly enhanced the apoptosis by INNO-406, even in INNO-406-less sensitive cells with Bcr-Abl point mutations except T315I mutation. In contrast, co-treatment with INNO-406 and other pharmacologic inducers of those BH3-only proteins, such as 17-allylaminogeldanamycin, an heat shock protein-90 inhibitor, or PS-341, a proteasome inhibitor, did not further increase the BH3-only protein levels or sensitize leukemic cells to INNO-406-induced apoptosis, suggesting a limit to how much expression levels of BH3-only proteins can be increased by anticancer agents. Thus, double-barrelled molecular targeting for Bcr-Abl-driven oncogenic signaling and the cell protection by antiapoptotic Bcl-2 family proteins may be the rational therapeutic approach for eradicating Ph+ leukemic cells.

Induction of autophagy in malignant rhabdoid tumor cells by the histone deacetylase inhibitor FK228 through AIF translocation

Malignant rhabdoid tumors (MRT) exhibit a very poor prognosis because of their resistance to chemotherapeutic agents and new therapies are needed for the treatment of this cancer. Here, we show that the histone deacetylase (HDAC) inhibitor FK228 (depsipeptide) has an antitumor effect on MRT cells both in vitro and in vivo. FK228 is a unique cyclic peptide and is among the most potent inhibitors of both Class I and Class II HDACs. FK228 inhibited proliferation and induced apoptosis in all MRT cell lines tested. Preincubation with the pancaspase inhibitor zVAD‐fmk did not completely rescue FK228‐induced cell death, although it did inhibit apoptosis. Transmission electron microscopy (TEM) showed that FK228 could stimulate MRT cells to undergo apoptosis, necrosis or autophagy. FK228 converted unconjugated microtubule‐associated protein light chain 3 (LC3‐I) to conjugated light chain 3 (LC3‐II) and induced localization of LC3 to autophagosomes. Apoptosis inducing factor (AIF), which plays a role in caspase‐independent cell death, translocated to the nucleus in response to FK228 treatment. Moreover, small interfering RNA (siRNA) targeting of AIF prevented the morphological changes associated with autophagy and redistribution of LC3 to autophagosomes. Disrupting autophagy with chloroquine treatment enhanced FK228‐induced cell death. In vivo, FK228 caused a reduction in tumor size and induced autophagy in tumor tissues. Using immunoelectron microscopy, we confirmed AIF translocation into the nucleus of FK228‐induced autophagic cells in vivo. Thus, FK228 is a novel candidate for an antitumor agent for MRT cells.

The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemias

Bcr-Abl tyrosine kinase (TK) inhibitors are promising therapeutic agents for Bcr-Abl-positive (Bcr-Abl+) leukemias. Although they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we demonstrated that INNO-406, a second-generation Bcr-Abl TK inhibitor, induces programmed cell death (PCD) in chronic myelogenous leukemia (CML) cell lines through both caspase-mediated and caspase-independent pathways. The latter pathways include caspase-independent apoptosis (CIA) and necrosis-like cell death (CIND), and the cell lines varied regarding which mechanism was elicited upon INNO-406 treatment. We also observed that the propensity toward CIA or CIND in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity. Cells that undergo CIND have a high apoptosome activity potential whereas cells that undergo CIA tend to have a lower potential. Moreover, we found that INNO-406 promotes autophagy. When autophagy was inhibited with chloroquine or gene knockdown of beclin1 by shRNA, INNO-406-induced cell death was enhanced, which indicates that the autophagic response of the tumor cells is protective. These findings suggest new insights into the biology and therapy of Bcr-Abl+ leukemias.

Administration of PLK-1 small interfering RNA with atelocollagen prevents the growth of liver metastases of lung cancer

Liver metastasis is one of the most important prognostic factors in lung cancer patients. However, current therapies are not sufficient. RNA interference provides us a powerful and promising approach for treating human diseases including cancers. Herein, we investigated the in vitro effects of PLK-1 small interfering RNA (siRNA) on human lung cancer cell lines and the in vivo usage of PLK-1 siRNA with atelocollagen as a drug delivery system in a murine liver metastasis model of lung cancer. PLK-1 was overexpressed in cell lines and in cancerous tissues from lung cancer patients. PLK-1 siRNA treatment inhibited growth and induced apoptosis in a concentration-dependent manner. To verify in vivo efficacy, we confirmed that atelocollagen was a useful drug delivery system in our model of implanted luciferase-labeled A549LUC cells by detecting reduced bioluminescence after an i.v. injection of luciferase GL3 siRNA/atelocollagen. PLK-1 siRNA/atelocollagen was also successfully transfected into cells and inhibited the progression of metastases. This study shows the efficacy of i.v. administration of PLK-1 siRNA/atelocollagen for liver metastases of lung cancer. We believe siRNA therapy will be a powerful and promising strategy against advanced lung cancer. [Mol Cancer Ther 2008;7(9):2904–12]

β-Catenin Small Interfering RNA Successfully Suppressed Progression of Multiple Myeloma in a Mouse Model

Purpose: β-catenin is the downstream effector of the Wnt signaling pathway, and it regulates cell proliferation. β-catenin overexpression correlates positively with prognosis in several types of malignancies. We herein assessed its effects on growth of multiple myeloma cells using a xenograft model. Experimental Design: We first investigated the expression of β-catenin in multiple myeloma cell lines and multiple myeloma cells obtained from patients. Next, we investigated the growth inhibitory effects of β-catenin small interfering RNA on the growth of multiple myeloma cells in vivo. Six-week-old male BALB/c nu/nu mice were inoculated s.c. in the right flank with 5 × 106 RPMI8226 cells, followed by s.c. injections of β-catenin small interfering RNA, scramble small interfering RNA, or PBS/atelocollagen complex twice a week for a total of eight injections. Results: Significantly higher levels of β-catenin expression were observed in multiple myeloma cell lines and in samples from patients with multiple myeloma than those found in mononuclear cells obtained from healthy volunteers. In in vivo experiments, no inhibitory effects were observed following treatment with scramble small interfering RNA or PBS/atelocollagen complexes, whereas treatment with β-catenin small interfering RNA/atelocollagen complex significantly inhibited growth of multiple myeloma tumors (P < 0.05). Conclusions: β-catenin small interfering RNA treatment inhibited the growth of multiple myeloma tumors in a xenograft model. To our knowledge, this is the first report showing that the treatment with β-catenin small interfering RNA produces an inhibitory effects on growth of hematologic malignancies in vivo. Because treatment with β-catenin small interfering RNA inhibited growth of multiple myeloma cells, β-catenin is the attractive novel target for treating multiple myeloma.

Comparison of Human Herpes Virus 8 Related Primary Effusion Lymphoma with Human Herpes Virus 8 Unrelated Primary Effusion Lymphoma-Like Lymphoma on the Basis of HIV: Report of 2 Cases and Review of 212 Cases in the Literature

Background: Primary lymphomatous effusion is a rare lymphoma that arises in the body cavity and has a peculiar proliferative form, lacking a tumor. This primary lymphomatous effusion includes human herpes virus 8 (HHV8)-related primary effusion lymphoma (PEL) and HHV8-unrelated PEL-like lymphoma. We attempted to clarify the nature of the primary lymphomatous effusion. Methods: Using ‘PEL’ and ‘body cavity-based lymphoma’ (BCBL) as key words, reports written in English were collected from PubMed. Primary lymphomatous effusion was defined as BCBL with primary effusion and without tumor at onset. Adding our 2 PEL-like lymphoma cases, each case was studied as to the patients’ and lymphomas’ characteristics, therapy and survival time. Moreover, each item was compared among four groups according to the presence of HHV8 and HIV. Results: In 214 cases investigated, there was no difference in proliferation, but an apparent difference in age, gender, phenotype, effectiveness and prognosis among the four groups. Conclusions: Both PEL and PEL-like lymphoma are thought to be characterized by a peculiar proliferation, regardless of the presence of HHV8. Dividing PEL or PEL-like lymphoma into two subgroups on the basis of HIV presentation might also be appropriate.

Anti-myeloma effect of homoharringtonine with concomitant targeting of the myeloma-promoting molecules, Mcl-1, XIAP, and β-catenin

Since a variety of cell intrinsic and extrinsic molecular abnormalities cooperatively promote tumor formation in multiple myeloma (MM), therapeutic approaches that concomitantly target more than one molecule are increasingly attractive. We herein demonstrate the anti-myeloma effect of a cephalotaxus alkaloid, homoharringtonine (HHT), an inhibitor of protein synthesis, through the induction of apoptosis. HHT significantly reduced Mcl-1, a crucial protein involved in myeloma cell survival, in all three myeloma cell lines examined, whereas certain BH3-only proteins, such as Bim, Bik, and Puma, remained unchanged following HHT treatment, and their expression levels depended on the cell type. HHT also reduced the levels of c-FLIPL/S, activated caspase-8, and induced active truncated-Bid. Thus, HHT-induced apoptosis appears to be mediated via both intrinsic and extrinsic apoptosis pathways, and the resultant imbalance between BH3-only proteins and Mcl-1 may be pivotal for apoptosis by HHT. In addition, HHT treatment resulted in reduced levels of β-catenin and XIAP proteins, which also contribute to disease progression and resistance to chemotherapy in MM. In combination, HHT enhanced the effects of melphalan, bortezomib, and ABT-737. These results suggest that HHT could constitute an attractive option for MM treatment though its ability to simultaneously target multiple tumor-promoting molecules.

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.

Osteoclasts are involved in the maintenance of dormant leukemic cells

Osteoclasts (OCs) are specialized cells for the resorption of bone matrix that have also been recently reported to be involved in the mobilization of hematopoietic progenitor cells. When Ba/F3 cells expressing wild-type bcr-abl were co-cultured with osteoblasts (OBs), OCs, and bone slices, their proliferation was significantly suppressed, and the Ki-67 negative population, which is believed to be in G(0) phase, was increased. The results of our in vitro experiments suggest that OCs could be involved in the maintenance of dormant leukemic cells in the bone marrow (BM) microenvironment through the release of soluble factors, one of which could be TGF-beta.