Hifzur R. Siddique

Role of BMI1, a Stem Cell Factor, in Cancer Recurrence and Chemoresistance: Preclinical and Clinical Evidences†‡§

There is increasing evidence that a variety of cancers arise from transformation of normal stem cells to cancer stem cells (CSCs). CSCs are thought to sustain cancer progression, invasion, metastasis, and recurrence after therapy. Reports suggest that CSCs are highly resistant to conventional therapy. Emerging evidences show that the chemoresistance of CSCs are in part due to the activation of B cell‐specific Moloney murine leukemia virus integration site 1 (BMI1), a stem cell factor, and a polycomb group family member. BMI1 is reported to regulate the proliferation activity of normal, stem, and progenitor cells. BMI1 plays a role in cell cycle, cell immortalization, and senescence. Numerous studies demonstrate that BMI1, which is upregulated in a variety of cancers, has a positive correlation with clinical grade/stage and poor prognosis. Although evidences are in support of the role of BMI1 as a factor in chemoresistance displayed by CSCs, its mechanism of action is not fully understood. In this review, we provide summary of evidences (with mechanism of action established) suggesting the significance of BMI1 in chemoresistance and recurrence of CSCs. STEM CELLS 2012;30:372–378

Beneficial health effects of lupeol triterpene: a review of preclinical studies.

Since ancient times, natural products have been used as remedies to treat human diseases. Lupeol, a phytosterol and triterpene, is widely found in edible fruits, and vegetables. Extensive research over the last three decades has revealed several important pharmacological activities of lupeol. Various in vitro and preclinical animal studies suggest that lupeol has a potential to act as an anti-inflammatory, anti-microbial, anti-protozoal, anti-proliferative, anti-invasive, anti-angiogenic and cholesterol lowering agent. Employing various in vitro and in vivo models, lupeol has also been tested for its therapeutic efficiency against conditions including wound healing, diabetes, cardiovascular disease, kidney disease, and arthritis. Lupeol has been found to be pharmacologically effective in treating various diseases under preclinical settings (in animal models) irrespective of varying routes of administration viz; topical, oral, intra-peritoneal and intravenous. It is noteworthy that lupeol has been reported to selectively target diseased and unhealthy human cells, while sparing normal and healthy cells. Published studies provide evidence that lupeol modulates the expression or activity of several molecules such as cytokines IL-2, IL4, IL5, ILβ, proteases, α-glucosidase, cFLIP, Bcl-2 and NFκB. This minireview discusses in detail the preclinical studies conducted with lupeol and provides an insight into its mechanisms of action.

S100A4 calcium-binding protein is key player in tumor progression and metastasis: preclinical and clinical evidence

The fatality of cancer is mainly bestowed to the property of otherwise benign tumor cells to become malignant and invade surrounding tissues by circumventing normal tissue barriers through a process called metastasis. S100A4 which is a member of the S100 family of calcium-binding proteins has been shown to be able to activate and integrate pathways both intracellular and extracellular to generate a phenotypic response characteristic of cancer metastasis. A large number of studies have shown an increased expression level of S100A4 in various types of cancers. However, its implications in cancer metastasis in terms of whether an increased expression of S100A4 is a causal factor for metastasis or just another after effect of several other physiological and molecular changes in the body resulting from metastasis are not clear. Here we describe the emerging preclinical and clinical evidences implicating S100A4 protein, in both its forms (intracellular and extracellular) in the process of tumorigenesis and metastasis in humans. Based on studies utilizing S100A4 as a metastasis biomarker and molecular target for therapies such as gene therapy, we suggest that S100A4 has emerged as a promising molecule to be tested for anticancer drugs. This review provides an insight in the (1) molecular mechanisms through which S100A4 drives the tumorigenesis and metastasis and (2) developments made in the direction of evaluating S100A4 as a cancer biomarker and drug target.

Lupeol, a Novel Androgen Receptor Inhibitor: Implications in Prostate Cancer Therapy

Purpose: Conventional therapies to treat prostate cancer (CaP) of androgen-dependent phenotype (ADPC) and castration-resistant phenotype (CRPC) are deficient in outcome which has necessitated a need to identify those agents that could target AR for both disease types. We provide mechanism-based evidence that lupeol (Lup-20(29)-en-3b-ol) is a potent inhibitor of androgen receptor (AR) in vitro and in vivo. Experimental Design: Normal prostate epithelial cell (RWPE-1), LAPC4 (wild functional AR/ADPC), LNCaP (mutant functional/AR/ADPC), and C4-2b (mutant functional/AR/CRPC) cells were used to test the anti-AR activity of lupeol. Cells grown under androgen-rich environment and treated with lupeol were tested for proliferation, AR transcriptional activity, AR competitive ligand binding, AR–DNA binding, and AR–ARE/target gene binding. Furthermore, in silico molecular modeling for lupeol–AR binding was done. Athymic mice bearing C4-2b and LNCaP cell–originated tumors were treated intraperitoneally with lupeol (40 mg/kg; 3 times/wk) and tumor growth and surrogate biomarkers were evaluated. To assess bioavailability, lupeol serum levels were measured. Results: Lupeol significantly inhibited R1881 (androgen analogue) induced (i) transcriptional activity of AR and (ii) expression of PSA. Lupeol (i) competed antagonistically with androgen for AR, (ii) blocked the binding of AR to AR-responsive genes including PSA, TIPARP, SGK, and IL-6, and (iii) inhibited the recruitment of RNA Pol II to target genes. Lupeol sensitized CRPC cells to antihormone therapy. High-performance liquid chromatography analysis showed that lupeol is bioavailable to mice. Lupeol inhibited the tumorigenicity of both ADPC and CRPC cells in animals. Serum and tumor tissues exhibited reduced PSA levels. Conclusion: Lupeol, an effective AR inhibitor, could be developed as a potential agent to treat human CaP. Clin Cancer Res; 17(16); 5379–91. ©2011 AACR.

Differential Effects of Genistein on Prostate Cancer Cells Depend on Mutational Status of the Androgen Receptor

Blocking the androgen receptor (AR) activity is the main goal of therapies for advanced prostate cancer (PCa). However, relapse with a more aggressive, hormone refractory PCa arises, which harbors restored AR activity. One mechanism of such reactivation occurs through acquisition of AR mutations that enable its activation by various steroidal and non-steroidal structures. Thus, natural and chemical compounds that contribute to inappropriate (androgen-independent) activation of the AR become an area of intensive research. Here, we demonstrate that genistein, a soy phytoestrogen binds to both the wild and the Thr877Ala (T877A) mutant types of AR competitively with androgen, nevertheless, it exerts a pleiotropic effect on PCa cell proliferation and AR activity depending on the mutational status of the AR. Genistein inhibited, in a dose-dependent way, cell proliferation and AR nuclear localization and expression in LAPC-4 cells that have wild AR. However, in LNCaP cells that express the T877A mutant AR, genistein induced a biphasic effect where physiological doses (0.5-5 µmol/L) stimulated cell growth and increased AR expression and transcriptional activity, and higher doses induced inhibitory effects. Similar biphasic results were achieved in PC-3 cells transfected with AR mutants; T877A, W741C and H874Y. These findings suggest that genistein, at physiological concentrations, potentially act as an agonist and activate the mutant AR that can be present in advanced PCa after androgen ablation therapy.

Comparative toxic potential of market formulation of two organophosphate pesticides in transgenic Drosophila melanogaster (hsp70-lacZ)

This study investigated the working hypothesis that two widely used organophosphate pesticides; Nuvan and Dimecron, exert toxic effects in Drosophila. Transgenic D. melanogaster (hsp70-lacZ) was used as a model for assaying stress gene expression and AchE activity as an endpoint for toxicity and also to evaluate whether stress gene expression is sufficient to protect against toxic insult of the chemicals and to prevent tissue damage. The study was extended to investigate the effect of the pesticides on the life cycle and reproduction of the organism. The study showed that Nuvan affected emergence of the exposed flies more drastically than Dimecron and the effect was lethal at the highest tested concentration (0.075 ppm). While Nuvan at 0.0075 and 0.015 ppm concentrations affected reproduction of the flies significantly, the effect of Dimecron was significant only at 0.015 and 0.075 ppm. Nuvan-exposed third-instar larvae exhibited a 1.2-fold to 1.5-fold greater hsp70 expression compared to Dimecron at concentrations ranging from 0.0075 to 0.075 ppm following 12 and 18 h exposure. While maximum expression of hsp70 was observed in Nuvan-exposed third-instar larval tissues following 18 h exposure at 0.075 ppm, Dimecron at the same dietary concentration induced a maximum expression of hsp70 following 24 h exposure. Further, concomitant with a significant induction of hsp70, significant inhibition of AchE was observed following chemical exposure and temperature shock. Concurrent with a significant decline in hsp70 expression in Nuvan-exposed larvae after 48 h at 0.075 ppm, tissue damage was evident. Dimecron-exposed larvae exhibited a plateau in hsp70 induction even after 48 h exposure and moderate tissue damage was observed in these larvae. The present study suggests that Nuvan is more cytotoxic than Dimecron in transgenic Drosophila melanogaster.

The S100A4 Oncoprotein Promotes Prostate Tumorigenesis in a Transgenic Mouse Model: Regulating NFκB through the RAGE Receptor.

S100A4, a calcium-binding protein, is known for its role in the metastatic spread of tumor cells, a late event of cancer disease. This is the first report showing that S100A4 is not merely a metastatic protein but also an oncoprotein that plays a critical role in the development of tumors. We earlier showed that S100A4 expression progressively increases in prostatic tissues with the advancement of prostate cancer (CaP) in TRAMP, an autochthonous mouse model. To study the functional significance of S100A4 in CaP, we generated a heterozygously deleted S100A4 (TRAMP/S100A4(+/-)) genotype by crossing TRAMP with S100A4(-/-) mice. TRAMP/S100A4(+/-) did not show a lethal phenotype, and transgenes were functional. As compared to age-matched TRAMP littermates, TRAMP/S100A4(+/-) mice exhibited 1) an increased tumor latency period (P < 0.001), 2) a 0% incidence of metastasis, and 3) reduced prostatic weights (P < 0.001). We generated S100A4-positive clones from S100A4-negative CaP cells and tested their potential. S100A4-positive tumors grew at a faster rate than S100A4-negative tumors in vitro and in a xenograft mouse model. The S100A4 protein exhibited growth factor-like properties in multimode (intracellular and extracellular) forms. We observed that 1) the growth-promoting effect of S100A4 is due to its activation of NFκB, 2) S100A4-deficient tumors exhibit reduced NFκB activity, 3) S100A4 regulates NFκB through the RAGE receptor, and 4) S100A4 and RAGE co-localize in prostatic tissues of mice. Keeping in view its growth-promoting role, we suggest that S100A4 qualifies as an excellent candidate to be exploited for therapeutic agents to treat CaP in humans.

Adverse effect of tannery waste leachates in transgenic Drosophila melanogaster: role of ROS in modulation of Hsp70, oxidative stress and apoptosis.

Leachate is a complex chemical mixture of chemicals produced as a result of leaching of solid wastes. The potential toxicity of leachates is a major environmental health concern. The present study evaluated the role of ROS in tannery leachates induced Hsp70 expression, antioxidant enzymes and apoptosis in Drosophila. Different concentrations (0.05–2.0%) of leachates prepared from tannery waste at different pH (7.00, 4.93 and 2.88) were mixed with Drosophila food and fed to the larvae for 2–48 h to examine the different stress and apoptotic markers. A concentration‐ and time‐dependent significant increase in Hsp70 expression, ROS generation, antioxidant enzymes activities and MDA content were observed in the exposed larvae. Activities of antioxidant enzymes were delayed compared with Hsp70 expression and MDA level in the exposed organisms. Apoptotic cell death was observed in the exposed larvae at higher concentrations concurrent with a significant regression in Hsp70 along with a higher level of ROS generation. A positive correlation drawn between ROS generation and apoptotic markers and a negative correlation between apoptotic markers and Hsp70 expression at these concentrations indicated the important role of ROS in the induction of cellular damage in the exposed organisms. There was a significant generation of ROS in the larvae exposed to 0.5% of leachates which did not interfere with the protection of their cells by Hsp70 and antioxidant enzymes. However, generation of significantly higher levels of ROS in the larvae exposed to 1.0% and 2.0% leachates may decrease Hsp70 expression thus leading to mitochondria‐mediated caspase‐dependent apoptotic cell death. Copyright

DNA damage induced by industrial solid waste leachates in Drosophila melanogaster: a mechanistic approach.

Genomic stability requires that error‐free genetic information be transmitted from generation to generation, a process that is dependent upon efficient DNA repair. Industrial leachates which contain mixtures of diverse chemicals are a major environmental concern. The interaction between these chemicals may have synergistic, antagonistic, or simply additive effects on biological systems. In the present study, the Comet assay was used to measure the DNA damage produced by leachates of solid wastes from flashlight battery, pigment, and tanning factories in the midgut cells and brain ganglia of Drosophila melanogaster mutants deficient in DNA repair proteins. Larvae were allowed to feed for 48 or 72 hr on diets containing 0.1, 0.5, and 2.0% (v/v) of the leachates. Physicochemical analysis run on the solid wastes, leachates, and treated larvae detected elevated levels of heavy metals. Leachates produced significantly greater levels of DNA damage in mutant strains mei41 (deficient in cell cycle check point protein), mus201 (deficient in excision repair protein), mus308 (deficient in postreplication repair protein), and rad54 (deficient in double strand break repair protein) than in the OregonR+ wild‐type strain. Larvae of the ligaseIV mutant (deficient in double strand break repair protein) were hypersensitive only to the pigment plant waste leachate. Conversely, the dnase2 mutant (deficient in protein responsible for degrading fragmented DNA) was more sensitive to DNA damage induction from the flashlight battery and tannery waste leachates. Our data demonstrate that repair of DNA damage in organisms exposed to leachates is dependent upon several DNA repair proteins, indicative of the involvement of multiple overlapping repair pathways. The study further suggests the usefulness of the Comet assay for studying the mechanisms of DNA repair in Drosophila. Environ. Mol. Mutagen., 2008.

BMI1 polycomb group protein acts as a master switch for growth and death of tumor cells: regulates TCF4-transcriptional factor-induced BCL2 signaling.

For advanced prostate cancer (CaP), the progression of tumors to the state of chemoresistance and paucity of knowledge about the mechanism of chemoresistance are major stumbling blocks in the management of this disease. Here, we provide compelling evidence that BMI1 polycomb group protein and a stem cell factor plays a crucial role in determining the fate of tumors vis-à-vis chemotherapy. We show that progressive increase in the levels of BMI1 occurs during the progression of CaP disease in humans. We show that BMI1-rich tumor cells are non-responsive to chemotherapy whereas BMI1-silenced tumor cells are responsive to therapy. By employing microarray, ChIP, immunoblot and Luciferase reporter assays, we identified a unique mechanism through which BMI1 rescues tumor cells from chemotherapy. We found that BMI1 regulates (i) activity of TCF4 transcriptional factor and (ii) binding of TCF4 to the promoter region of anti-apoptotic BCL2 gene. Notably, an increased TCF4 occupancy on BCL2 gene was observed in prostatic tissues exhibiting high BMI1 levels. Using tumor cells other than CaP, we also showed that regulation of TCF4-mediated BCL2 by BMI1 is universal. It is noteworthy that forced expression of BMI1 was observed to drive normal cells to hyperproliferative mode. We show that targeting BMI1 improves the outcome of docetaxel therapy in animal models bearing chemoresistant prostatic tumors. We suggest that BMI1 could be exploited as a potential molecular target for therapeutics to treat chemoresistant tumors.