Aslamuzzaman Kazi

Loss of Bif-1 Suppresses Bax/Bak Conformational Change and Mitochondrial Apoptosis

ABSTRACT Bif-1, a member of the endophilin B protein family, interacts with Bax and promotes interleukin-3 withdrawal-induced Bax conformational change and apoptosis when overexpressed in FL5.12 cells. Here, we provide evidence that Bif-1 plays a regulatory role in apoptotic activation of not only Bax but also Bak and appears to be involved in suppression of tumorigenesis. Inhibition of endogenous Bif-1 expression in HeLa cells by RNA interference abrogated the conformational change of Bax and Bak, cytochrome c release, and caspase 3 activation induced by various intrinsic death signals. Similar results were obtained in Bif-1 knockout mouse embryonic fibroblasts. While Bif-1 did not directly interact with Bak, it heterodimerized with Bax on mitochondria in intact cells, and this interaction was enhanced by apoptosis induction and preceded the Bax conformational change. Moreover, suppression of Bif-1 expression was associated with an enhanced ability of HeLa cells to form colonies in soft agar and tumors in nude mice. Taken together, these findings support the notion that Bif-1 is an important component of the mitochondrial pathway for apoptosis as a novel Bax/Bak activator, and loss of this proapoptotic molecule may contribute to tumorigenesis.

Inhibition of the proteasome activity, a novel mechanism associated with the tumor cell apoptosis-inducing ability of genistein.

Epidemiological studies have suggested that increased soy consumption is associated with reduced cancer occurrence. Genistein, a soy isoflavone, has been reported to inhibit the growth of human tumor cells although the involved molecular mechanisms are not clearly defined. Here we report that genistein inhibits the proteasomal chymotrypsin-like activity in vitro and in vivo. Computational docking studies suggest that the interaction of genistein with the proteasomal beta 5 subunit is responsible for inhibition of the chymotrypsin-like activity. Inhibition of the proteasome by genistein in prostate cancer LNCaP and breast cancer MCF-7 cells is associated with accumulation of ubiquitinated proteins and three known proteasome target proteins, the cyclin-dependent kinase inhibitor p27(Kip1), inhibitor of nuclear factor-kappa B (I kappa B-alpha), and the pro-apoptotic protein Bax. Genistein-mediated proteasome inhibition was accompanied by induction of apoptosis in these solid tumor cells. Finally, genistein induced proteasome inhibition and apoptosis selectively in simian virus 40-transformed human fibroblasts, but not in their parental normal counterpart. Our results suggest that the proteasome is a potential target of genistein in human tumor cells and that inhibition of the proteasome activity by genistein might contribute to its cancer-preventive properties.

Phase II Study of the Mitogen-Activated Protein Kinase 1/2 Inhibitor Selumetinib in Patients With Advanced Hepatocellular Carcinoma

PURPOSE Hepatocellular carcinoma (HCC) is a common and deadly malignancy with few systemic therapy options. The RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-related kinase (ERK) pathway is activated in approximately 50% to 60% of HCCs and represents a potential target for therapy. Selumetinib is an orally available inhibitor of MEK tyrosine kinase activity. PATIENTS AND METHODS Patients with locally advanced or metastatic HCC who had not been treated with prior systemic therapy were enrolled on to the study. Patients were treated with selumetinib at its recommended phase II dose of 100 mg twice per day continuously. Cycle length was 21 days. Imaging was performed every two cycles. Biopsies were obtained at baseline and at steady-state in a subset of patients, and pharmacokinetic (PK) analysis was performed on all patients. Results Nineteen patients were enrolled, 17 of whom were evaluable for response. Most (82%) had Child-Pugh A cirrhosis. Toxicity was in line with other studies of selumetinib in noncirrhotic patients. PK parameters were also comparable to those in noncirrhotic patients. No radiographic response was observed in this group, and the study was stopped at the interim analysis. Of 11 patients with elevated α-fetoprotein, three (27%) had decreases of 50% or more. Median time to progression was 8 weeks. Inhibition of ERK phosphorylation was demonstrated by Western blotting. CONCLUSION In this study of selumetinib for patients with HCC, no radiographic responses were seen and time to progression was short, which suggests minimal single-agent activity despite evidence of suppression of target activation.

The BH3 α-Helical Mimic BH3-M6 Disrupts Bcl-XL, Bcl-2, and MCL-1 Protein-Protein Interactions with Bax, Bak, Bad, or Bim and Induces Apoptosis in a Bax- and Bim-dependent Manner

A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-XL, and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-XL and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-XL, Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-XL/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-XL, Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.

Targeted Inhibition of Farnesyltransferase in Locally Advanced Breast Cancer: A Phase I and II Trial of Tipifarnib Plus Dose-Dense Doxorubicin and Cyclophosphamide

PURPOSE To determine the recommended phase II dose (RPTD) of the farnesyltransferase (FTase) inhibitor tipifarnib when combined with doxorubicin and cyclophosphamide (AC) in patients with advanced breast cancer, the pathologic complete response (pCR) rate after preoperative treatment with four cycles of the combination in locally advanced breast cancer (LABC), and the effect of tipifarnib on primary tumor FTase enzyme activity in vivo. PATIENTS AND METHODS Thirty-two patients with metastatic breast cancer (n = 11) or LABC (n = 21) received AC (doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2) administered intravenously on day 1 plus tipifarnib (100, 200, or 300 mg bid for 6 to 14 days) without (n = 2) or with (n = 30) granulocyte colony-stimulating factor (G-CSF) for up to four cycles. Patients with LABC underwent surgery after up to four cycles of the combination. RESULTS When combined with AC every 2 weeks plus G-CSF, the RPTD of tipifarnib was 200 mg bid administered on days 2 to 7. Seven (33%) of 21 patients (95% CI, 15% to 55%) with LABC treated with up to four cycles of the combination at the RPTD had a pCR in the breast at surgery. The five patients had serial biopsies that demonstrated at least 50% FTase enzyme inhibition in the primary tumor (median, 100%; range, 55% to 100%) after tipifarnib. CONCLUSION Tipifarnib may be safely combined with dose-dense AC using a dose and schedule that significantly inhibits FTase enzyme activity in human breast cancer in vivo and may enhance the pCR rate after four cycles of preoperative dose-dense AC.

Green tea and tea polyphenols in cancer prevention.

The cancer-preventive effects of green tea and its main constituent (-)-epigallocatechin gallate [(-)-EGCG] are widely supported by results from epidemiological, cell culture, animal and clinical studies in the recent decade. In vitro cell culture studies show that tea polyphenols potently induce apoptotic cell death and cell cycle arrest in tumor cells but not in their normal cell counterparts. Green tea polyphenols affect several signal transduction pathways, including growth factor-mediated, the mitogen-activated protein kinase (MAPK)-dependent, and ubiquitin/proteasome degradation pathways. Epidemiological studies have suggested that the consumption of green tea lowers the risk of cancer. Various animal studies have revealed that treatment by green tea inhibits tumor incidence and multiplicity in different organ sites such as skin, lung, liver, stomach, mammary gland and colon. Phase I and II clinical trials were carried out recently to explore the anticancer effects of green tea in patients with cancer. At this time, more mechanistic research, animal studies, and clinical trials are necessary to further evaluate the role of green tea in cancer prevention.

SYNTHETIC PERACETATE TEA POLYPHENOLS AS POTENT PROTEASOME INHIBITORS AND APOPTOSIS INDUCERS IN HUMAN CANCER CELLS

It has been suggested that proteasome activity is essential for tumor cell proliferation and drug resistance development. We have previously shown that natural and synthetic ester bond-containing tea polyphenols are selective inhibitors of the chymotrypsin-like activity of the proteasome. The most abundant catechin in green tea is (-)-epigallocatechin-3-gallate [(-)-EGCG], which has been found by many laboratories to exhibit the most potent anticancer activity. We have reported that (-)-EGCG is also the most effective proteasome inhibitor among all the natural green tea catechins tested. Unfortunately, (-)-EGCG is very unstable in neutral and alkaline conditions. In an attempt to increase the stability and thus the efficacy, we synthesized several (-)-EGCG analogs with acetyl protected -OH groups as prodrugs. Here we report, for the first time, that these acetylated synthetic tea analogs are much more potent than natural (-)-EGCG in inhibiting the proteasome in cultured tumor cells. Consistently, these protected analogs showed much higher potency than (-)-EGCG to inhibit proliferation and transforming activity and to induce apoptosis in human leukemic, prostate, breast, and simian virus 40-transformed cells. Additionally, these protected analogs had greatly reduced effects on human normal and non-transformed cells. Therefore, these peracetate protected tea polyphenols are more efficacious than (-)-EGCG and possess great potential to be developed into novel anticancer drugs. Identification of the cytosolic metabolite(s) of peracetate-protected polyphenols in cultured tumor cells and examination of their in vivo tumor growth-inhibitory activity are currently underway in our laboratory.

Phase II Trial of Tipifarnib plus Neoadjuvant Doxorubicin-Cyclophosphamide in Patients with Clinical Stage IIB-IIIC Breast Cancer

Purpose: Tipifarnib is a farnesyl transferase (FTase) inhibitor that has activity in metastatic breast cancer and enhances the efficacy of cytotoxic agents in preclinical models. We evaluated the biological effects of tipifarnib in primary breast cancers in vivo, whether adding tipifarnib to preoperative chemotherapy increased the pathologic complete response rate (pCR) at surgery, and determined whether biomarkers predictive of pCR could be identified. Experimental Design: Forty-four patients with stage IIB-IIIC breast cancer received up to four cycles of neoadjuvant doxorubicin-cyclophosphamide (AC) every 2 weeks plus tipifarnib and filgrastim followed by surgery. Enzymatic assays measuring FTase activity and Western blotting for phospho (p)-signal transducer and activator of transcription 3 (STAT3), phospho-extracellular signal-regulated kinase, p-AKT, and p27 were done in 11 patients who agreed to optional tissue biopsies before therapy and 2 hours after the final dose of tipifarnib during the first cycle, and predictive biomarkers were evaluated by immunohistochemistry in 33 patients. The trial was powered to detect an improvement in breast pCR rate of 10% or less expected for AC alone to 25% for AC-tipifarnib (α = 0.05, β = 0.10). Results: Eleven patients had a breast pCR (25%; 95% confidence interval, 13-40%). FTase enzyme activity decreased in all patients (median, 91%; range, 24-100%) and p-STAT3 expression decreased in 7 of 9 (77%) patients. Low tumor Ki-67 expression (below the median of 60%) at baseline was significantly associated with resistance to therapy (P = 0.01). Conclusion: Tipifarnib inhibits FTase activity in human breast tumors in vivo, is associated with down-regulation of p-STAT3, and enhances the breast pCR rate, thus meriting further evaluation.

Discovery of a novel proteasome inhibitor selective for cancer cells over non-transformed cells

Numerous proteins controlling cell cycle progression, apoptosis, and angiogenesis are degraded by the ubiquitin/proteasome system, which has become the subject for intense investigations for cancer therapeutics. Therefore, we used in silico and experimental approaches to screen compounds from the NCI chemical libraries for inhibitors against the chymotrypsin-like (CT-L) activity of the proteasome and discovered PI-083. Molecular docking indicates that PI-083 interacts with the Thr21, Gly47 and Ala49 residues of the β5 subunit and Asp114 of the β6 subunit of the proteasome. PI-083 inhibits CT-L activity and cell proliferation and induces apoptosis selectively in cancer cells (ovarian T80-Hras, pancreatic C7-Kras and breast MCF-7) as compared to their normal/immortalized counterparts (T80, C7 and MCF-10A, respectively). In contrast, Bortezomib, the only proteasome inhibitor approved by the Food and Drug Administration (FDA), did not exhibit this selectivity for cancer over non-transformed cells. In addition, in all cancer cells tested, including Multiple Myeloma (MM), breast, pancreatic, ovarian, lung, prostate cancer cell lines as well as fresh MM cells from patients, PI-083 required less time than Bortezomib to induce its antitumor effects. Furthermore, in nude mouse xenografts in vivo, PI-083, but not Bortezomib, suppressed the growth of human breast and lung tumors. Finally, following in vivo treatment of mice, PI-083 inhibited tumor, but not hepatic liver CT-L activity, whereas Bortezomib inhibited both tumor and liver CT-L activities. These results suggest that PI-083 is more selective for cancer cells and may have broader antitumor activity and therefore warrants further advanced preclinical studies.

Randomized, Placebo-Controlled Trial of Green Tea Catechins for Prostate Cancer Prevention

Preclinical, epidemiologic, and prior clinical trial data suggest that green tea catechins (GTC) may reduce prostate cancer risk. We conducted a placebo-controlled, randomized clinical trial of Polyphenon E (PolyE), a proprietary mixture of GTCs, containing 400 mg (−)-epigallocatechin-3-gallate (EGCG) per day, in 97 men with high-grade prostatic intraepithelial neoplasia (HGPIN) and/or atypical small acinar proliferation (ASAP). The primary study endpoint was a comparison of the cumulative one-year prostate cancer rates on the two study arms. No differences in the number of prostate cancer cases were observed: 5 of 49 (PolyE) versus 9 of 48 (placebo), P = 0.25. A secondary endpoint comparing the cumulative rate of prostate cancer plus ASAP among men with HGPIN without ASAP at baseline, revealed a decrease in this composite endpoint: 3 of 26 (PolyE) versus 10 of 25 (placebo), P < 0.024. This finding was driven by a decrease in ASAP diagnoses on the Poly E (0/26) compared with the placebo arm (5/25). A decrease in serum prostate-specific antigen (PSA) was observed on the PolyE arm [−0.87 ng/mL; 95% confidence intervals (CI), −1.66 to −0.09]. Adverse events related to the study agent did not significantly differ between the two study groups. Daily intake of a standardized, decaffeinated catechin mixture containing 400 mg EGCG per day for 1 year accumulated in plasma and was well tolerated but did not reduce the likelihood of prostate cancer in men with baseline HGPIN or ASAP. Cancer Prev Res; 8(10); 879–87. ©2015 AACR.