Bilal Bin Hafeez

Introducing Nanochemoprevention as a Novel Approach for Cancer Control: Proof of Principle with Green Tea Polyphenol Epigallocatechin-3-Gallate

Chemoprevention, especially through the use of naturally occurring phytochemicals capable of impeding the process of one or more steps of carcinogenesis process, is a promising approach for cancer management. Despite promising results in preclinical settings, its applicability to humans has met with limited success largely due to inefficient systemic delivery and bioavailability of promising chemopreventive agents. Here, we introduce the concept of nanochemoprevention, which uses nanotechnology for enhancing the outcome of chemoprevention. We encapsulated green tea polyphenol epigallocatechin-3-gallate (EGCG) in polylactic acid-polyethylene glycol nanoparticles and observed that encapsulated EGCG retains its biological effectiveness with over 10-fold dose advantage for exerting its proapoptotic and angiogenesis inhibitory effects, critically important determinants of chemopreventive effects of EGCG in both in vitro and in vivo systems. Thus, this study could serve as a basis for the use of nanoparticle-mediated delivery to enhance bioavailability and limit any unwanted toxicity of chemopreventive agents, such as EGCG.

Green tea polyphenol EGCG sensitizes human prostate carcinoma LNCaP cells to TRAIL-mediated apoptosis and synergistically inhibits biomarkers associated with angiogenesis and metastasis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) is a promising candidate for cancer therapy, however, emergence of drug resistance limits its potential use. Here, we report for the first time that epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent of green tea, sensitizes TRAIL-resistant LNCaP cells to TRAIL-mediated apoptosis through modulation of intrinsic and extrinsic apoptotic pathways. When combined with EGCG, Apo2L/TRAIL exhibited enhanced apoptotic activity in LNCaP cells characterized by three major molecular events. First, apoptosis induction was accompanied by the upregulation of poly(ADP-ribose) polymerase cleavage and modulation of pro- and antiapoptotic Bcl2 family of proteins. A synergistic inhibition of inhibitors of apoptosis with concomitant increase in caspase cleavage was observed. Second, pretreatment of cells with EGCG resulted in modulation of death-inducing signaling cascade complex involving DR4/TRAIL R1, Fas-associated death domain and FLICE-inhibitory protein proteins. Last, we observed a synergistic inhibition in the invasion and migration of LNCaP cells. This effect was observed to be mediated through inhibition in the protein expression of vascular endothelial growth factor, uPA and angiopoietin 1 and 2. Further, the activity and protein expression of MMP-2, -3 and -9 and upregulation of TIMP1 in cells treated with a combination of EGCG and TRAIL was observed. These data might have implications for developing new strategies aimed at eliminating prostate cancer cells resistant to TRAIL.

S100A4 accelerates tumorigenesis and invasion of human prostate cancer through the transcriptional regulation of matrix metalloproteinase 9

We previously showed that the calcium-binding protein S100A4 is overexpressed during the progression of prostate cancer (CaP) in humans and in the TRAMP (transgenic adenocarcinoma of the mouse prostate) mouse model. We tested a hypothesis that the S100A4 gene plays a role in the invasiveness of human CaP and may be associated with its metastatic spread. We observed that siRNA-mediated suppression of the S100A4 gene significantly reduced the proliferative and invasive capability of the highly invasive CaP cells PC-3. We evaluated the mechanism through which the S100A4 gene controls invasiveness of cells by using a macroarray containing 96 well characterized metastatic genes. We found that matrix metalloproteinase 9 (MMP-9) and its tissue inhibitor (TIMP-1) were highly responsive to S100A4 gene suppression. Furthermore, S100A4 suppression significantly reduced the expression and proteolytic activity of MMP-9. By employing an MMP-9-promoter reporter, we observed a significant reduction in the transcriptional activation of the MMP-9 gene in S100A4-siRNA-transfected cells. Cells overexpressing the S100A4 gene (when transfected with pcDNA3.1-S100A4 plasmid) also significantly expressed MMP-9 and TIMP-1 genes with increased proteolytic activity of MMP-9 concomitant to increased transcriptional activation of the MMP-9 gene. S100A4-siRNA-transfected cells exhibited a reduced rate of tumor growth under in vivo conditions. Our data demonstrate that the S100A4 gene controls the invasive potential of human CaP cells through regulation of MMP-9 and that this association may contribute to metastasis of CaP cells. We suggest that S100A4 could be used as a biomarker for CaP progression and a novel therapeutic or chemopreventive target for human CaP treatment.

A Dietary Anthocyanidin Delphinidin Induces Apoptosis of Human Prostate Cancer PC3 Cells In vitro and In vivo: Involvement of Nuclear Factor-κB Signaling

Delphinidin, a major anthocyanidin present in many pigmented fruits and vegetables, possesses antioxidant, anti-inflammatory, and antiangiogenic properties. In this study, we provide evidence that it could be developed as a novel agent against human prostate cancer (PCa). We observed that delphinidin treatment to human PCa LNCaP, C4-2, 22Rnu1, and PC3 cells resulted in a dose-dependent inhibition of cell growth without having any substantial effect on normal human prostate epithelial cells. We selected PC3 cells as a test model system because of their highly aggressive proliferative nature. Delphinidin treatment of cells resulted in a dose-dependent induction of apoptosis and arrest of cells in G(2)-M phase. This induction of apoptosis seems to be mediated via activation of caspases because N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluromethylketone significantly reduced apoptosis induced by delphinidin. We also observed that delphinidin treatment of cells resulted in a dose-dependent decrease in (a) phosphorylation of IkappaB kinase gamma (NEMO), (b) phosphorylation of nuclear factor-kappaB (NF-kappaB) inhibitory protein IkappaBalpha, (c) phosphorylation of NF-kappaB/p65 at Ser(536) and NF-kappaB/p50 at Ser(529), (d) NF-kappaB/p65 nuclear translocation, and (e) NF-kappaB DNA binding activity. Delphinidin administration (2 mg, i.p. thrice weekly) to athymic nude mice implanted with PC3 cells resulted in a significant inhibition of tumor growth. Analysis of tumors from delphinidin-treated mice showed significant decrease in the expression of NF-kappaB/p65, Bcl2, Ki67, and PCNA. Taken together, our data suggest that delphinidin could be developed as an agent against human PCa.

Green tea polyphenol EGCG blunts androgen receptor function in prostate cancer

Androgen deprivation therapy is the major treatment for advanced prostate cancer (PCa). However, it is a temporary remission, and the patients almost inevitably develop hormone refractory prostate cancer (HRPC). HRPC is almost incurable, although most HRPC cells still express androgen receptor (AR) and depend on the AR for growth, making AR a prime drug target. Here, we provide evidence that epigallocatechin‐3‐gallate (EGCG), the major polyphenol in green tea, is a direct antagonist of androgen action. In silico modeling and FRET‐based competition assay showed that EGCG physically interacts with the ligandbinding domain of AR by replacing a high‐affinity labeled ligand (IC50 0.4 µM). The functional consequence of this interaction was a decrease in AR‐mediated transcriptional activation, which was due to EGCG mediated inhibition of interdomain N‐C termini interaction of AR. Treatment with EGCG also repressed the transcriptional activation by a hotspot mutant AR (T877A) expressed ectopically as well as the endogenous AR mutant. As the physiological consequence of AR antagonism, EGCG repressed R1881‐induced PCa cell growth. In a xenograft model, EGCG was found to inhibit AR nuclear translocation and protein expression. We also observed a significant down‐regulation of androgen‐regulated miRNA‐21 and up‐regulation of a tumor suppressor, miRNA‐330, in tumors of mice treated with EGCG. Taken together, we provide evidence that EGCG functionally antagonizes androgen action at multiple levels, resulting in inhibition of PCa growth.—Siddiqui, I. A., Asim, M., Hafeez, B. B., Adhami, V. M., Tarapore, R. S., Mukhtar, H. Green tea polyphenol EGCG blunts androgen receptor function in prostate cancer. FASEB J. 25, 1198–1207 (2011). www.fasebj.org

Targeted knockdown of Notch1 inhibits invasion of human prostate cancer cells concomitant with inhibition of matrix metalloproteinase-9 and urokinase plasminogen activator.

Purpose:Notch, a type 1 transmembrane protein, plays a key role in the development of many tissues and organ types. Aberrant Notch signaling, found in a wide variety of human cancers, contributes to tumor development. Because Notch1 was found to be overexpressed in prostate cancer (PCa) cells and human PCa tissue, we therefore tested our hypothesis that overexpression of Notch1 in PCa promotes tumor invasion. Experimental Design:Notch1 expression was evaluated in human PCa cells and human PCa tissues. PCa cells were transiently transfected with Notch1-specific small interfering RNAs in concentrations ranging from 30 to 120 nmol/L and subsequently evaluated for effects on invasion and expression analysis for molecules involved in invasion. Results: Small interfering RNA–mediated knockdown of Notch1 in PC3 and 22Rν1 PCa cells dramatically decreased their invasion. Focused cDNA array revealed that Notch1 knockdown resulted in significant reduction in the expression of urokinase plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9) gene transcripts. These data were further verified by reverse transcription-PCR, real-time reverse transcription-PCR, and immunoblot analysis. Knockdown of Notch1 was also observed to significantly reduce the mRNA expression and protein levels of uPA and its receptor uPAR. A significant reduction in MMP9 expression in Notch1 knockdown cells suggested a role for Notch1 in augmenting MMP9 transcription. Conclusions: Our data show the involvement of Notch1 in human PCa invasion and that silencing of Notch1 inhibits invasion of human PCa cells by inhibiting the expression of MMP9 and uPA. Thus, targeting of Notch1 could be an effective therapeutic approach against PCa.

Lupeol Inhibits Growth of Highly Aggressive Human Metastatic Melanoma Cells In vitro and In vivo by Inducing Apoptosis

Purpose: Poor prognosis of metastatic melanoma mandates the development of novel strategies for its treatment and prevention. In this study, the effect of lupeol, a diet-based triterpene, was determined on the growth and tumorigenicity of human melanoma cells in vitro and in vivo. Experimental Design: Normal human melanocytes, and human metastatic (451Lu) and nonmetastatic (WM35) cells were treated with lupeol; its effect on growth, proliferation, and apoptosis were evaluated. Further athymic nude mice bearing 451Lu cell–originated tumors were administered with lupeol thrice a week, and its effect on tumor growth and surrogate biomarkers was evaluated. Results: Lupeol significantly decreased the viability of 451Lu and WM35 melanoma cells but had only a marginal effect on normal human melanocyte cells at similar doses. Lupeol treatment of 451Lu cells caused (a) G1-S phase cell cycle arrest and apoptosis; (b) down-regulation of Bcl2 and up-regulation of Bax; (c) activation of caspase-3 and induction of poly(ADP)ribose polymerase cleavage; (d) decreased expression of cyclin D1, cyclin D2, and cdk2; and (e) increased expression of p21 protein. Next, lupeol significantly reduced 451Lu tumor growth in athymic nude mice and modulated the expression of proliferation markers, apoptotic markers, and cell cycle regulatory molecules in tumor xenografts. Conclusion: Our findings showed the anticancer efficacy of lupeol with mechanistic rationale against metastatic human melanoma cells. We suggest that lupeol, alone or as an adjuvant to current therapies, could be useful for the management of human melanoma.

Effective Prostate Cancer Chemopreventive Intervention with Green Tea Polyphenols in the TRAMP Model Depends on the Stage of the Disease

Purpose: We have shown previously that oral feeding of green tea polyphenols (GTP) to transgenic adenocarcinoma of the mouse prostate mice in a purely chemopreventive setting significantly inhibits prostate cancer development. To translate this to a human situation, the present study was designed to identify the stage of prostate cancer that is most vulnerable to chemopreventive intervention by GTP. Experimental Design: GTP infusion (0.1% in drinking water) to transgenic adenocarcinoma of the mouse prostate was initiated at ages representing different stage of the disease: (a) 6 weeks (group 1, normal prostate), (b) 12 weeks (group 2, prostatic intraepithelial neoplasia), (c) 18 weeks (group 3, well-differentiated adenocarcinoma), and (b) 28 weeks (group 4, moderately differentiated adenocarcinoma). At age 32 weeks, subsets of animals were evaluated by magnetic resonance imaging, ultrasound, and prostate weight and for serum insulin-like growth factor (IGF)-I/IGF binding protein-3 and IGF signaling. Results: Tumor-free survival was extended to 38 weeks (P < 0.001) in group 1, 31 weeks (P < 0.01) in group 2, and 24 weeks (P < 0.05) in group 3 compared with 19 weeks in water-fed controls. Median life expectancy was 68 weeks in group 1, 63 weeks in group 2, 56 weeks in group 3, and 51 weeks in group 4 compared with 42 weeks in the control mice. IGF-I and its downstream targets including phosphatidylinositol 3-kinase, pAkt, and phosphorylated extracellular signal-regulated kinase were significantly inhibited only when intervention was initiated early when prostatic intraepithelial neoplasia lesions were common. Conclusions: Our studies indicate that chemopreventive potential of GTP decreases with advancing stage of the disease and underscore the need to design appropriate chemoprevention clinical trails.

Fisetin, a natural flavonoid, targets chemoresistant human pancreatic cancer AsPC-1 cells through DR3-mediated inhibition of NF-κB.

Death receptors of the tumor necrosis factor (TNF) receptor super family have been implicated in constitutive activation of nuclear factor‐kappa B (NF‐κB) in pancreatic cancer (PaC) cells. In this study, we demonstrate that fisetin, a natural flavonoid, induces apoptosis and inhibits invasion of chemoresistant PaC AsPC‐1 cells through suppression of DR3‐mediated NF‐κB activation. Fisetin treatment resulted in dose‐dependent inhibition of PaC cell growth and cell proliferation with concomitant induction of apoptosis. A cDNA array analysis revealed that fisetin modulates expression of more than 20 genes at transcription level with maximum decrease observed in DR3 expression and a parallel increase observed in the expression levels of IκBα, an NF‐κB inhibitor. Down‐regulation of DR3 in PaC cells was found to down regulate activated pNF‐κB/p65, pIkBα/β kinases (pIKKs), MMP9 and XIAP that mostly impart chemoresistance in PaC. Immunoblotting and EMSA analysis showed a marked decrease in pNF‐κB and NF‐κB DNA binding activity, respectively, with modest decrease in NF‐κB promoter activity and significant decrease in MMP9 promoter activity with fisetin treatment. Importantly, consistent with these findings, we further found that transient down‐regulation of DR3 by RNA interference significantly augmented fisetin induced changes in cell proliferation, cell invasion and apoptosis paralleled with decrease in pNF‐κB, pIKKα/β, MMP9, XIAP and NF‐κB DNA binding activity. Blocking of DR3 receptor with an extra cellular domain blocking antibody demonstrated similar effects. These data provide evidence that fisetin could provide a biological rationale for treatment of pancreatic cancer or as an adjuvant with conventional therapeutic regimens.

Protein kinase Cvarepsilon mediates Stat3Ser727 phosphorylation, Stat3-regulated gene expression, and cell invasion in various human cancer cell lines through integration with MAPK cascade (RAF-1, MEK1/2, and ERK1/2).

Protein kinase C epsilon (PKCɛ), a novel calcium-independent PKC isoform, has been shown to be a transforming oncogene. PKCɛ-mediated oncogenic activity is linked to its ability to promote cell survival. However, the mechanisms by which PKCɛ signals cell survival remain elusive. We found that signal transducers and activators of transcription 3 (Stat3), which is constitutively activated in a wide variety of human cancers, is a protein partner of PKCɛ. Stat3 has two conserved amino-acid (Tyr705 and Ser727) residues, which are phosphorylated during Stat3 activation. PKCɛ interacts with Stat3α isoform, which has Ser727, and not with Stat3β isoform, which lacks Ser727. PKCɛ–Stat3 interaction and Stat3Ser727 phosphorylation was initially observed during induction of squamous cell carcinomas and in prostate cancer. Now we present that (1) PKCɛ physically interacts with Stat3α isoform in various human cancer cells: skin melanomas (MeWo and WM266-4), gliomas (T98G and MO59K), bladder (RT-4 and UM-UC-3), colon (Caco-2), lung (H1650), pancreatic (PANC-1), and breast (MCF-7 and MDA:MB-231); (2) inhibition of PKCɛ expression using specific siRNA inhibits Stat3Ser727 phosphorylation, Stat3-DNA binding, Stat3-regulated gene expression as well as cell invasion; and (3) PKCɛ mediates Stat3Ser727 phosphorylation through integration with the MAPK cascade (RAF-1, MEK1/2, and ERK1/2). The results indicate that PKCɛ-mediated Stat3Ser727 phosphorylation is essential for constitutive activation of Stat3 and cell invasion in various human cancers.