Fazlul H. Sarkar

Multi-targeted therapy of cancer by genistein

Soy isoflavones have been identified as dietary components having an important role in reducing the incidence of breast and prostate cancers in Asian countries. Genistein, the predominant isoflavone found in soy products, has been shown to inhibit the carcinogenesis in animal models. There is a growing body of experimental evidence showing that the inhibition of human cancer cell growth by genistein is mediated via the modulation of genes that are related to the control of cell cycle and apoptosis. It has been shown that genistein inhibits the activation of NF-kappaB and Akt signaling pathways, both of which are known to maintain a homeostatic balance between cell survival and apoptosis. Moreover, genistein antagonizes estrogen- and androgen-mediated signaling pathways in the processes of carcinogenesis. Furthermore, genistein has been found to have antioxidant properties, and shown to be a potent inhibitor of angiogenesis and metastasis. Taken together, both in vivo and in vitro studies have clearly shown that genistein, one of the major soy isoflavones is a promising agent for cancer chemoprevention and further suggest that it could be an adjunct to cancer therapy by virtue of its effects on reversing radioresistance and chemoresistance. In this review, we attempt to provide evidence for these preventive and therapeutic effects of genistein in a succinct manner highlighting comprehensive state-of-the-art knowledge regarding its multi-targeted biological and molecular effects in cancer cells.

Exosomes in Cancer Development, Metastasis and Drug Resistance: A Comprehensive Review

Trafficking of biological material across membranes is an evolutionary conserved mechanism and is part of any normal cell homeostasis. Such transport is composed of active, passive, export through microparticles, and vesicular transport (exosomes) that collectively maintain proper compartmentalization of important micro- and macromolecules. In pathological states, such as cancer, aberrant activity of the export machinery results in expulsion of a number of key proteins and microRNAs resulting in their misexpression. Exosome-mediated expulsion of intracellular drugs could be another barrier in the proper action of most of the commonly used therapeutics, targeted agents, and their intracellular metabolites. Over the last decade, a number of studies have revealed that exosomes cross-talk and/or influence major tumor-related pathways, such as hypoxia-driven epithelial-to-mesenchymal transition, cancer stemness, angiogenesis, and metastasis involving many cell types within the tumor microenvironment. Emerging evidence suggests that exosome-secreted proteins can also propel fibroblast growth, resulting in desmoplastic reaction, a major barrier in effective cancer drug delivery. This comprehensive review highlights the advancements in the understanding of the biology of exosomes secretions and the consequence on cancer drug resistance. We propose that the successful combination of cancer treatments to tackle exosome-mediated drug resistance requires an interdisciplinary understanding of these cellular exclusion mechanisms, and how secreted biomolecules are involved in cellular cross-talk within the tumor microenvironment.

Pathologically and biologically distinct types of epithelium in intraductal papillary mucinous neoplasms: Delineation of an intestinal pathway of carcinogenesis in the pancreas

Although general characteristics of intraductal papillary mucinous neoplasms (IPMNs) and their delineation from other pancreatic tumors have been well established, several issues regarding their biology and management remain unresolved. It has been noted briefly by us and other authors that there are different types of papillae in IPMNs; however, their frequency, biologic significance, and clinical relevance are unknown. In this study, the association of different papillary patterns with clinical, pathologic, and biologic parameters was studied in 74 IPMNs, and the expression profile of CDX2 (a specific marker and one of the key determinants of intestinal “programming,” and a tumor suppressor) was determined immunohistochemically in addition to MUC1 (a marker of an “aggressive” phenotype in pancreatic neoplasia) and MUC2 (“intestinal type mucin,” a marker of the “indolent” phenotype, and a tumor suppressor). The patterns of papillae identified and their association with these parameters were as follows: 1) The intestinal-type (Yonezawa’s dark-cell type), similar to villous adenomas, was seen in 26 of 74 (35%) cases. The majority harbored carcinoma in situ (85%) or borderline atypia (15%). They tended to be large (mean, 5.5 cm). Most expressed CDX2 (95%) and MUC2 (92%) but not MUC1 (8%). This type was more commonly associated with colloid-type invasion (14 of 16 invasive carcinomas were of colloid type). 2) The pancreatobiliary type, characterized by arborizing papillae lined by cuboidal cells resembling papillary neoplasms of the biliary tract, was present in 22% of the cases. These were mostly graded as carcinoma in situ (94%); they rarely expressed CDX2 (6%) or MUC2 (19%) but often showed MUC1 labeling (44%). This pattern was more commonly associated with the tubular type of invasive carcinoma and had a slight tendency for a more aggressive clinical course. 3) The null type was characterized by abundant apical mucin and basally located nuclei, similar to the gastric foveolar epithelium. Thirty-one percent of IPMNs had this type of papillae, but this pattern was also present in the background of other IPMNs and in the cystic components of most cases as well. Most pure null-type IPMNs were devoid of complexity and consequently classified as adenoma (48%). They tended to be small (mean, 2.6 cm), were often negative for CDX2, MUC1, and MUC2, and were rarely associated with invasive carcinoma. 4) Some IPMNs (12%) exhibited features that were difficult to classify, and 2 cases had a mixture of pancreatobiliary and intestinal types of papillae. In conclusion, IPMNs include pathologically and biologically distinct epithelial patterns. CDX2 and MUC2 expression is relatively specific for the intestinal type papillae, confirming that these IPMNs indeed exhibit intestinal differentiation. Their close association with colloid carcinoma, which also shows consistent MUC2 and CDX2 expression, supports the existence of an intestinal pathway of carcinogenesis. This “metaplastic” pathway may reflect different genetic events in the development of these IPMNs, and the presence of intestinal differentiation may potentially be used in prognostication and stratification of patients into appropriate treatment categories.

Gemcitabine Sensitivity Can Be Induced in Pancreatic Cancer Cells through Modulation of miR-200 and miR-21 Expression by Curcumin or Its Analogue CDF

Curcumin induces cancer cell growth arrest and apoptosis in vitro, but its poor bioavailability in vivo limits its antitumor efficacy. We have previously evaluated the bioavailability of novel analogues of curcumin compared with curcumin, and we found that the analogue CDF exhibited greater systemic and pancreatic tissue bioavailability. In this study, we evaluated the effects of CDF or curcumin alone or in combination with gemcitabine on cell viability and apoptosis in gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer (PC) cell lines. Mechanistic investigations revealed a significant reduction in cell viability in CDF-treated cells compared with curcumin-treated cells, which were also associated with the induction of apoptosis, and these results were consistent with the downregulation of Akt, cyclooxygenase-2, prostaglandin E(2), vascular endothelial growth factor, and NF-kappaB DNA binding activity. We have also documented attenuated expression of miR-200 and increased expression of miR-21 (a signature of tumor aggressiveness) in gemcitabine-resistant cells relative to gemcitabine-sensitive cells. Interestingly, CDF treatment upregulated miR-200 expression and downregulated the expression of miR-21, and the downregulation of miR-21 resulted in the induction of PTEN. These results prompt further interest in CDF as a drug modality to improve treatment outcome of patients diagnosed with PC as a result of its greater bioavailability in pancreatic tissue.

Inactivation of Nuclear Factor κB by Soy Isoflavone Genistein Contributes to Increased Apoptosis Induced by Chemotherapeutic Agents in Human Cancer Cells

Cancer chemotherapeutic strategies commonly require multiple agents. However, use of multiple agents contributes to added toxicity resulting in poor treatment outcome. Thus, combination chemotherapy must be optimized to increase tumor response and at the same time lower its toxicity. Chemotherapeutic agents are known to induce nuclear factor kappaB (NF-kappaB) activity in tumor cells, resulting in lower cell killing and drug resistance. In contrast, genistein has been shown to inhibit the activity of NF-kappaB and the growth of various cancer cells without causing systemic toxicity. We therefore investigated whether the inactivation of NF-kappaB by genistein before treatment of various cancer cells with chemotherapeutic agents could lead to better tumor cell killing as tested by in vitro studies using gene transfections and also by animal studies. PC-3 (prostate), MDA-MB-231 (breast), H460 (lung), and BxPC-3 (pancreas) cancer cells were pretreated with 15 to 30 micromol/L genistein for 24 hours and then exposed to low doses of chemotherapeutic agents for an additional 48 to 72 hours. We found that 15 to 30 micromol/L genistein combined with 100 to 500 nmol/L cisplatin, 0.5 to 2 nmol/L docetaxel, or 50 ng/mL doxorubicin resulted in significantly greater inhibition of cell growth and induction of apoptosis compared with either agent alone. Moreover, we found that the NF-kappaB activity was significantly increased within 2 hours of cisplatin and docetaxel treatment and that the NF-kappaB inducing activity of these agents was completely abrogated in cells pretreated with genistein. These results were also supported, for the first time, by animal experiments, p65 cDNA transfection and p65 small interfering RNA studies, which clearly showed that a specific target (NF-kappaB) was affected in vivo. Collectively, our results clearly suggest that genistein pretreatment inactivates NF-kappaB and may contribute to increased growth inhibition and apoptosis induced by cisplatin, docetaxel, and doxorubicin in prostate, breast, lung, and pancreatic cancer cells. Theses results warrant carefully designed clinical studies investigating the combination of soy isoflavones and commonly used chemotherapeutic agents for the treatment of human cancers.

Soy Isoflavones and Cancer Prevention

Epidemiological studies have shown a significant difference in cancer incidence among different ethnic groups, which is believed to be partly attributed to dietary habits. The incidences of breast and prostate cancers are much higher in the United States and European countries compared with Asian countries such as Japan and China. One of the major differences in diet between these populations is that the Japanese and the Chinese consume a traditional diet high in soy products. Soy isoflavones have been identified as dietary components having an important role in reducing the incidence of breast and prostate cancers. Genistein, the predominant isoflavones found in soy, has been shown to inhibit the carcinogenesis in animal models. There are growing body of experimental evidence that show the inhibition of human cancer cells by genistein through the modulation of genes that are related to the control of cell cycle and apoptosis. Moreover, it has been shown that genistein inhibits the activation of NF-κB and Akt signaling pathways, both of which are known to maintain a homeostatic balance between cell survival and apoptosis. Genistein is commonly known as phytoestrogen, which targets estrogen- and androgen-mediated signaling pathways in the processes of carcinogenesis. Furthermore, genistein has been found to have antioxidant property, and shown to be a potent inhibitor of angiogenesis and metastasis. Taken together, both in vivo and in vitro studies have clearly shown that genistein, one of the major soy isoflavones, is a promising reagent for cancer chemoprevention and/or treatment. In this article, we attempt to provide evidence for these effects of genistein in a succinct manner to provide comprehensive state-of-the-art knowledge of the biological and molecular effects of the isoflavone genistein in cancer cells.

Down-regulation of Notch-1 Inhibits Invasion by Inactivation of Nuclear Factor-κB, Vascular Endothelial Growth Factor, and Matrix Metalloproteinase-9 in Pancreatic Cancer Cells

Notch signaling plays a critical role in the pathogenesis and progression of human malignancies but the precise role and mechanism of Notch-1 for tumor invasion remains unclear. In our earlier report, we showed that down-regulation of Notch-1 reduced nuclear factor-kappaB (NF-kappaB) DNA-binding activity and matrix metalloproteinase-9 (MMP-9) expression. Because NF-kappaB, VEGF, and MMPs are critically involved in the processes of tumor cell invasion and metastasis, we investigated the role and mechanism(s) by which Notch-1 down-regulation (using molecular approaches) may lead to the down-regulation of NF-kappaB, vascular endothelial growth factor (VEGF), and MMP-9, thereby inhibiting invasion of pancreatic cancer cells through Matrigel. We found that the down-regulation of Notch-1 by small interfering RNA decreased cell invasion, whereas Notch-1 overexpression by cDNA transfection led to increased tumor cell invasion. Consistent with these results, we found that the down-regulation of Notch-1 reduced NF-kappaB DNA-binding activity and VEGF expression. Down-regulation of Notch-1 also decreased not only MMP-9 mRNA and its protein expression but also inactivated the pro-MMP-9 protein to its active form. Taken together, we conclude that the down-regulation of Notch-1 could be an effective approach for the down-regulation and inactivation of NF-kappaB and its target genes, such as MMP-9 and VEGF expression, resulting in the inhibition of invasion and metastasis.

Mechanisms of Cancer Chemoprevention by Soy Isoflavone Genistein

Diet has been implicated to play an important role in cancers. Epidemiological studies have revealed that Asians, who consume a traditional diet high in soy products, have relatively low incidences of breast and prostate cancers, while the incidences are much higher in the Western world. Asians who immigrate to the United States and adopt a Western diet are at higher risks of breast and prostate cancers. Soy isoflavones have received much attention as dietary components having an important role in reducing breast and prostate cancers. Genistein, one of the predominant soy isoflavones, has been shown to inhibit the growth of cancer cells through the modulation of genes that are related to the homeostatic control of cell cycle and apoptosis. It has been found that genistein inhibits the activation of the nuclear transcription factor, NF-κB and Akt signaling pathway, both of which are known to maintain a balance between cell survival and programmed cell death (apoptosis). Genistein is known to have anti-oxidant property, and commonly known as phytoestrogen, which targets estrogen and androgen-mediated signaling pathway in the processes of carcinogenesis. Moreover, genistein is also found to be a potent inhibitor of angiogenesis and metastasis. Hence, significant advances have been made, both by in vitro and in vivo studies showing that genistein is a promising agent for cancer chemoprevention and/or treatment.

miR-200 Regulates PDGF-D-Mediated Epithelial-Mesenchymal Transition, Adhesion, and Invasion of Prostate Cancer Cells

MicroRNAs have been implicated in tumor progression. Recent studies have shown that the miR‐200 family regulates epithelial–mesenchymal transition (EMT) by targeting zinc‐finger E‐box binding homeobox 1 (ZEB1) and ZEB2. Emerging evidence from our laboratory and others suggests that the processes of EMT can be triggered by various growth factors, such as transforming growth factor β and platelet‐derived growth factor‐D (PDGF‐D). Moreover, we recently reported that overexpression of PDGF‐D in prostate cancer cells (PC3 PDGF‐D cells) leads to the acquisition of the EMT phenotype, and this model offers an opportunity for investigating the molecular interplay between PDGF‐D signaling and EMT. Here, we report, for the first time, significant downregulation of the miR‐200 family in PC3 PDGF‐D cells as well as in PC3 cells exposed to purified active PDGF‐D protein, resulting in the upregulation of ZEB1, ZEB2, and Snail2 expression. Interestingly, re‐expression of miR‐200b in PC3 PDGF‐D cells led to reversal of the EMT phenotype, which was associated with the downregulation of ZEB1, ZEB2, and Snail2 expression, and these results were consistent with greater expression levels of epithelial markers. Moreover, transfection of PC3 PDGF‐D cells with miR‐200b inhibited cell migration and invasion, with concomitant repression of cell adhesion to the culture surface and cell detachment. From these results, we conclude that PDGF‐D‐induced acquisition of the EMT phenotype in PC3 cells is, in part, a result of repression of miR‐200 and that any novel strategy by which miR‐200 could be upregulated would become a promising approach for the treatment of invasive prostate cancer. STEM CELLS 2009;27:1712–1721

Induction of apoptosis in breast cancer cells MDA-MB-231 by genistein.

Breast cancer is the most common cancer among American women, whereas Asian women, who consume a traditional diet high in soy products, have a relatively low incidence. Genistein is a prominent isoflavonoid in soy products and has been proposed as the agent responsible for lowering the rate of breast cancer in Asian women. We investigated the effects of genistein on cell growth and apoptosis-related gene expression in breast cancer cells MDA-MB-231. We found up-regulation of Bax and p21WAF1 expressions and down-regulation of Bcl-2 and p53 expression in genistein-treated cells. Furthermore, DNA ladder formation, CPP32 activation, and PARP cleavage were observed after treatment with genistein, indicating apoptotic cell deaths. Flow cytometry with 7-amino actinomycin D staining showed that the number of apoptotic cells increased with longer treatment of genistein. From these results, we conclude that genistein inhibits the growth of MDA-MB-231 breast cancer cells, regulates the expression of apoptosis-related genes, and induces apoptosis through a p53-independent pathway. The up-regulation of Bax and p21WAF1 may be the molecular mechanisms by which genistein induces apoptosis, however, further definitive studies are needed. These results suggest that genistein may be a potentially effective chemopreventive or therapeutic agent against breast cancer.