Previously, we showed that sulforaphane (SFN), a naturally occurring cancer chemopreventive agent, effectively inhibits proliferation of PC-3 human prostate cancer cells by causing caspase-9-and caspase-8-mediated apoptosis. Here, we demonstrate that SFN treatment causes an irreversible arrest in the G 2 /M phase of the cell cycle. Cell cycle arrest induced by SFN was associated with a significant decrease in protein levels of cyclin B1, cell division cycle (Cdc) 25B, and Cdc25C, leading to accumulation of Tyr-15-phosphorylated (inactive) cyclin-dependent kinase 1. The SFN-induced decline in Cdc25C protein level was blocked in the presence of proteasome inhibitor lactacystin, but lactacystin did not confer protection against cell cycle arrest. Interestingly, SFN treatment also resulted in a rapid and sustained phosphorylation of Cdc25C at Ser-216, leading to its translocation from the nucleus to the cytoplasm because of increased binding with 14-3-3␤. Increased Ser-216 phosphorylation of Cdc25C upon treatment with SFN was the result of activation of checkpoint kinase 2 (Chk2), which was associated with Ser-1981 phosphorylation of ataxia telangiectasia-mutated, generation of reactive oxygen species, and Ser-139 phosphorylation of histone H2A.X, a sensitive marker for the presence of DNA double-strand breaks. Transient transfection of PC-3 cells with Chk2-specific small interfering RNA duplexes significantly attenuated SFN-induced G 2 /M arrest. HCT116 human colon cancer-derived Chk2 ؊/؊ cells were significantly more resistant to G 2 /M arrest by SFN compared with the wild type HCT116 cells. These findings indicate that Chk2-mediated phosphorylation of Cdc25C plays a major role in irreversible G 2 /M arrest by SFN. Activation of Chk2 in response to DNA damage is well documented, but the present study is the first published report to link Chk2 activation to cell cycle arrest by an isothiocyanate.Epidemiological studies have revealed an inverse correlation between the dietary intake of cruciferous vegetables and the risk for certain types of cancers, including prostate cancer (1-5). Laboratory studies indicate that the anticancer effect of cruciferous vegetables is caused by isothiocyanates that exist as thioglucoside conjugates (glucosinolates) in a variety of edible plants including broccoli, cabbage, watercress, and so forth (6 -9). Cruciferous vegetable-derived organic isothiocyanates are generated by hydrolytic cleavage of corresponding glucosinolates through catalytic mediation of myrosinases, which are released when the plant cells are damaged because of cutting or chewing (6 -9). Sulforaphane (SFN) 1 is one such isothiocyanate analog that has received a great deal of attention not only because it is present in high concentrations in certain varieties of broccoli but also because of its potent anticancer activity (10 -15). For example, oral administration of SFN (1-isothiocyanato-4-(methylsulfinyl)butane; CH 3 -SO-(CH 2 ) 4 -NϭCϭS)) caused a statistically significant reduction in 9,10-dimethyl-1,2-benzanthr...

Sulforaphane (SFN), a constituent of cruciferous vegetables, is highly effective in affording protection against chemically induced cancers in animal models. Here, we report that SFN inhibited proliferation of cultured PC-3 human prostate cancer cells by inducing apoptosis that was characterized by appearance of cells with sub-G0/G1 DNA content, formation of cytoplasmic histone associated DNA fragments and cleavage of poly(ADP-ribose)polymerase (PARP). SFN-induced apoptosis was associated with up-regulation of Bax, down-regulation of Bcl-2 and activation of caspases-3, -9 and -8. SFN-induced apoptosis, and cleavage of procaspase-3 and PARP were blocked upon pre-treatment of cells with pan caspase inhibitor z-VADfmk, and specific inhibitors of caspase-9 (z-LEHDfmk) and caspase-8 (z-IETDfmk) suggesting involvement of both caspase-9 and caspase-8 pathways in SFN-induced cell death. Oral administration of SFN (5.6 micro mol, 3 times/week) significantly inhibited growth of PC-3 xenografts in nude mice. For instance, 10 days after starting therapy, the average tumor volumes in control and SFN-treated mice were 170 +/- 13 and 80 +/- 14 mm3, respectively, reflecting a >50% reduction in tumor volume due to SFN administration. To the best of our knowledge, the present study is the first published report to document in vivo anticancer activity of SFN in a tumor xenograft model.

Summary Multiple Myeloma (MM) remains incurable despite novel therapies, suggesting the need for further identification of factors mediating tumorigenesis and drug resistance. Using both in vitro and in vivo MM xenograft models, we show that plasmacytoid dendritic cells (pDCs) in the bone marrow (BM) microenvironment both mediate immune deficiency characteristic of MM and promote MM cell growth, survival, and drug resistance. Microarray, cell signaling, cytokine profile and immunohistochemical analysis delineate the mechanisms mediating these sequelae. Although pDCs are resistant to novel therapies, targeting Toll-like Receptors with CpG ODNs both restores pDC immune function and abrogates pDC-induced MM cell growth. Our study therefore validates targeting pDC-MM interactions as a therapeutic strategy to overcome drug resistance in MM.

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Our recent study demonstrated that a novel proteasome inhibitor NPI-0052 triggers apoptosis in multiple myeloma (MM) cells, and importantly, that is distinct from bortezomib (Velcade) in its chemical structure, effects on proteasome activities, and mechanisms of action. Here, we demonstrate that combining NPI-0052 and bortezomb induces synergistic anti-MM activity both in vitro using MM cell lines or patient CD138(+) MM cells and in vivo in a human plasmacytoma xenograft mouse model. NPI-0052 plus bortezomib-induced synergistic apoptosis is associated with: (1) activation of caspase-8, caspase-9, caspase-3, and PARP; (2) induction of endoplasmic reticulum (ER) stress response and JNK; (3) inhibition of migration of MM cells and angiogenesis; (4) suppression of chymotrypsin-like (CT-L), caspase-like (C-L), and trypsin-like (T-L) proteolytic activities; and (5) blockade of NF-kappaB signaling. Studies in a xenograft model show that low dose combination of NPI-0052 and bortezomib is well tolerated and triggers synergistic inhibition of tumor growth and CT-L, C-L, and T-L proteasome activities in tumor cells. Immununostaining of MM tumors from NPI-0052 plus bortezomib-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Taken together, our study provides the preclinical rationale for clinical protocols evaluating bortezomib together with NPI-0052 to improve patient outcome in MM.

Bortezomib therapy has proven successful for the treatment of relapsed, relapsed/ refractory, and newly diagnosed multiple myeloma (MM). At present, bortezomib is available as an intravenous injection, and its prolonged treatment is associated with toxicity and development of drug resistance. Here we show that the novel proteasome inhibitor ONX 0912, a tripeptide epoxyketone, inhibits growth and induces apoptosis in MM cells resistant to conventional and bortezomib therapies. IntroductionThe Ubiquitin-Proteasome Signaling (UPS) pathway regulates normal cellular processes including cell cycle, DNA replication, transcription, and cell death via proteolysis of regulatory proteins. Alterations in UPS are linked to the pathogenesis of various human diseases, 1 and therefore, targeting UPS components such as proteasomes offers great promise as a novel therapeutic strategy. Bortezomib is the first-in-class proteasome inhibitor, approved by the Food and Drug Administration for the treatment of relapsed, relapsed/refractory, and newly diagnosed multiple myeloma (MM). [1][2][3][4][5] Although bortezomib therapy is a major advance, 3,4 it has been associated with possible off-target toxicities and the development of drug resistance. 6,7 On the heels of clinical success of bortezomib, many recent studies have focused on developing other proteasome inhibitors as therapeutics in cancer. In this context, recent reports demonstrated that carfilzomib (PR-171), a structural analog of the microbial natural product epoxomicin, triggers potent antitumor activity 8,9 associated with inhibition of chymotrypsin-like (CT-L) proteasome activity. 9-12 A Phase II clinical trial of carfilzomib in relapsed-refractory MM has shown promising single agent activity, including responses in patients relapsed from or refractory to bortezomib therapy. 13 However, like bortezomib, carfilzomib is also administered intravenously in patients. More recently, an orally bioavailable analog of carfilzomib, ONX 0912, was discovered during a medicinal chemistry effort using tripeptide epoxyketones. 14 Importantly, the clinical applicability of ONX 0912 will allow for enhanced dosing flexibility and patient convenience.In the present study, we examined the antitumor activity of ONX 0912 using MM cell lines and primary patient cells, as well as animal models. ONX 0912, like carfilzomib and bortezomib, triggers marked anti-MM activity associated with inhibition of proteasome CT-L activity. In vivo studies, using 2 distinct human MM xenograft mouse models, showed that ONX 0912 is well tolerated, inhibits tumor growth, and prolongs survival in mice. The combination of ONX 0912 with bortezomib, lenalidomide, pan-histone deacetylase (HDAC) inhibitor MS-275 or dexamethasone induces synergistic/additive anti-MM activity. Current bortezomib or carfilzomib therapy requires intravenous administration, whereas ONX 0912 is orally bioactive. Overall, our preclinical data provide the framework for clinical trials of ONX 0912, either alone or in combination with other anti-M...

A role of dietary bioactive components in bladder cancer prevention is biologically plausible because most substances or metabolites are excreted through the urinary tract and are consequently in direct contact with the mucosa of the bladder. We first determined antigrowth activity of genistein against poorly differentiated 253J B-V human bladder cancer cells in vitro. Genistein inhibited the cell growth in a time-and dosedependent manner via G 2 -M arrest, down-regulation of nuclear factor KB (NF-KB), and induction of apoptosis. We also evaluated both genistin, which is a natural form of genistein, and the isoflavone-rich soy phytochemical concentrate (SPC) on the growth and metastasis of 253J B-V tumors in an orthotopic tumor model. Mice treated with genistin and SPC had reduced final tumor weights by 56% (P < 0.05) and 52% (P < 0.05), respectively, associated with induction of tumor cell apoptosis and inhibition of tumor angiogenesis in vivo. In addition, SPC treatment, but not genistin treatment, significantly inhibited lung metastases by 95% (P < 0.01) associated with significant down-regulation of NF-KB expression in tumor tissues and reduction of circulating insulin-like growth factor-I levels, suggesting that SPC may contain other bioactive ingredients that have antimetastatic activity. The results from our studies suggest that further clinical investigation should be warranted to apply soy phytochemicals, such as SPC, as a potent prevention regimen for bladder cancer progression. This orthotopic human bladder tumor model also provides a clinically relevant experimental tool for assessing potential preventive activity of other dietary components against bladder tumor growth and metastasis.

SummaryPR-924 is an LMP-7-selective tripeptide epoxyketone proteasome inhibitor that covalently modifies proteasomal N-terminal threonine active sites. In the present study, we show that PR-924 inhibits growth and triggers apoptosis in multiple myeloma (MM) cell lines and primary patient MM cells, without significantly affecting normal peripheral blood mononuclear cells. PR-924-induced apoptosis in MM cells is associated with activation of caspase-3, caspase-8, caspase-9, BID, PARP and cytochrome-c release. In vivo administration of PR-924 inhibits tumour growth in human plasmacytoma xenografts. Results from SCID-hu model show a significant reduction in the shIL-6R levels in mice treated with PR-924 versus vehicle-control. PR-924 treatment was well tolerated as evidenced by the lack of weight loss. Importantly, treatment of tumour-bearing mice with PR-924, but not vehicle alone, prolonged survival. Our preclinical findings therefore validate immunoproteasome LMP-7 subunit as a novel therapeutic target in MM.