Synthesis and Photophysical Properties of Tumor-Targeted Water-Soluble BODIPY Photosensitizers for Photodynamic Therapy.

The synthesis of three water-soluble lactose-modified 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-based photosensitizers with tumor-targeting capabilities is reported, including an investigation into their photodynamic therapeutic activity on three distinct cancer cell lines (human hepatoma Huh7, cervical cancer HeLa, and breast cancer MCF-7 cell lines). The halogenated BODIPY dyes exhibited a decreased fluorescence quantum yield compared to their non-halogenated counterpart, and facilitated the efficient generation of singlet oxygen species. The synthesized dyes exhibited low cytotoxicities in the dark and high photodynamic therapeutic capabilities against the treated cancer cell lines following irradiation at 530 nm. Moreover, the incorporation of lactose moieties led to an enhanced cellular uptake of the BODIPY dyes. Collectively, the results presented herein provide promising insights for the development of photodynamic therapeutic agents for cancer treatment.

Inclusion complexes of cysteinyl ß-cyclodextrin with baicalein restore collagen synthesis in fibroblast cells following ultraviolet exposure.

Baicalein, a bioactive flavonoid, has poor water solubility, thereby limiting its use in a wide range of biological applications. In the present study, we used inclusion complexes of cysteinyl ß-cyclodextrin (ß-CD) with baicalein to enhance the stability and solubility of baicalein in aqueous solution. We examined the effects of inclusion complexes of cysteinyl ß-CD on collagen synthesis following ultraviolet (UV) irradiation, as well as the mechanisms underlying its effects. Our findings demonstrated that baicalein significantly restored collagen synthesis in the UV-exposed human fibroblast Hs68 cells. In addition, synthetic cysteine functionalized ß-CDs were found to promote baicalein-induced collagen synthesis. Inclusion complexes of cysteinyl ß-CDs with baicalein significantly upregulated the protein expression of type I collagen and activated the transcription of type I, II, and III collagen. Inclusion complexes of cysteinyl ß-CDs with baicalein also downregulated matrix metalloproteinase -1 and -3, and α-smooth muscle actin expression. In addition, inclusion complexes of cysteinyl ß-CDs with baicalein attenuated the expression of caveolin-1, but this treatment enhanced the UV-induced phosphorylation of Smad in the transforming growth factor-ß pathway. These results suggested that the newly synthesized derivative of CD can be used as a complexing agent to enhance the bioavailability of flavonoids such as baicalein, especially in restoring collagen synthesis.

Withaferin A inhibits expression of ataxia telangiectasia and Rad3-related kinase and enhances sensitivity of human breast cancer cells to cisplatin.

Ataxia telangiectasia and Rad3-related (ATR) is a serine/threonine-specific kinase that plays an important role in the maintenance of genomic integrity. In this study, we investigated the role of ATR in cell-cycle arrest by withaferin A (WA), a cancer preventative steroidal lactone derived from Withania somnifera plant abundant in India and surrounding countries. The WA treatment decreased the viability of MCF-7, MDA-MB-231, and SUM159 cells. Exposure of breast cancer cells to WA also resulted in suppression of protein level as well as phosphorylation of ATR and its downstream effector kinase (checkpoint kinase 1; CHK1). Both transcriptional and posttranscriptional mechanisms were involved in the WA-mediated downregulation of ATR protein. Downregulation of ATR protein expression resulting from WA exposure was not attenuated by overexpression of manganese superoxide dismutase. In contrast, the overexpression of CHK1 attenuated WA-mediated G2 /M arrest and augmented S10 phosphorylation of histone H3, a marker of mitotic arrest. The protein level of ATR was lowered by about 50% in breast tumors of WA-treated mouse mammary tumor virus-neu mice when compared with vehicle-treated controls but the difference was not significant due to small sample size. WA treatment sensitized MDA-MB-231 and SUM159 cells to growth inhibition and apoptosis induction by cisplatin. Cisplatin treatment resulted in increased autophosphorylation of ATR (T1989) and CHK1 (S345) phosphorylation that was markedly suppressed in the presence of WA. These results indicate that WA is an inhibitor of ATR in human breast cancer cells.

3,3'-Diindolylmethane Inhibits TNF-α- and TGF-ß-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells.

Epithelial-mesenchymal transition (EMT) is the initial event required by cancer cells. Thus, inhibition of the EMT process could have potential benefits for preventing the spread of cancers. The phytochemicals have been reported to have inhibitory activity against the EMT process in breast cancers, but the mechanism behind this effect has not been fully elucidated. 3,3'-Diindolylmethane (DIM) is a major indole derived from bioactive compounds in cruciferous vegetables. In this study, we examined the effects of DIM cotreatment together with TNF-α/TGF-ß on the EMT process as well as the mechanisms underlying its effects on human breast cancer cells. DIM significantly enhanced the mRNA and protein expression of E-cadherin and occludin in MCF-7 cells. The protein expression levels of E-cadherin and occludin in MCF-7 cells were significantly decreased after TNF-α/TGF-ß treatment alone, but these effects were reversed by the DIM co-treatment. Furthermore, DIM with TNF-α/TGF-ß co-treatment attenuated the phosphorylation of Smad2/3 and ERK1/2 proteins. DIM significantly inhibited the TNF-α/TGF-ß-induced migration of breast cancer cells. Taken together, the results indicated that DIM effectively suppressed EMT processes through the inhibition of TNF-α/TGF-ß-associated signaling pathways in breast cancer cells. Thus, DIM may be a novel preventive and/or therapeutic approach for the treatment of breast cancers.

Peptidyl-prolyl cis/trans isomerase Pin1 regulates withaferin A-mediated cell cycle arrest in human breast cancer cells.

We have reported previously that withaferin A (WA) prevents breast cancer development in mouse mammary tumor virus-neu (MMTV-neu) transgenic mice, but the mechanism is not fully understood. Unbiased proteomics of the mammary tumors from control- and WA-treated MMTV-neu mice revealed downregulation of peptidyl-prolyl cis/trans isomerase (Pin1) protein by WA administration. The present study extends these findings to elucidate the role of Pin1 in cancer chemopreventive mechanisms of WA. The mammary tumor level of Pin1 protein was lower by about 55% in WA-treated rats exposed to N-methyl-N-nitrosourea, compared to control. Exposure of MCF-7 and SK-BR-3 human breast cancer cells to WA resulted in downregulation of Pin1 protein. Ectopic expression of Pin1 attenuated G2 and/or mitotic arrest resulting from WA treatment in both MCF-7 and SK-BR-3 cells. WA-induced apoptosis was increased by Pin1 overexpression in MCF-7 cells but not in the SK-BR-3 cell line. In addition, molecular docking followed by mass spectrometry indicated covalent interaction of WA with cysteine 113 of Pin1. Overexpression of Pin1C113A mutant failed to attenuate WA-induced mitotic arrest or apoptosis in the MCF-7 cells. Furthermore, antibody array revealed upregulation of proapoptotic insulin-like growth factor binding proteins (IGFBPs), including IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6, in Pin1 overexpressing MCF-7 cells following WA treatment when compared to empty vector transfected control cells. These data support a crucial role of the Pin1 for mitotic arrest and apoptosis signaling by WA at least in the MCF-7 cells.

Properties of Heparinoids Premixed with Tumor-Derived Extracellular Vesicles.

Tumor-derived exosomes are bound and internalized to organ-specific cells, affecting metastasis. Heparan sulfate proteoglycans mediate the interaction between cells and exosomes. Exosome transfer to the recipient cell can be competitively blocked by heparinoids, because heparin is structurally similar to heparan sulfate. It is hypothesized that there may be structural requirements of heparinoids to attenuate the cellular uptake and metastatic activity of tumor-derived exosomes. Here, we compared the properties of unfractionated heparin (UFH), glycol-split UFH, low-molecular-weight heparin (LMWH), glycol-split LMWH, and ultra-LMWH premixed with A549-derived exosomes. Uptake of A549-derived exosomes (0.1 mg/mL) into BEAS-2B cells was significantly blocked by 0.4 mg/mL of heparinoids. Heparinoids attenuated migration of BEAS-2B cells stimulated by A549-derived exosomes. Glycol-split LMWH with no antifactor Xa activity exhibited the strongest antimigratory effects than other heparinoids. Thus, heparinoids with proper molecular weight and structure can inhibit tumor-derived exosomes, not proportionally to the anticoagulant activity.

Withaferin A inhibits in vivo growth of breast cancer cells accelerated by Notch2 knockdown.

The present study offers novel insights into the molecular circuitry of accelerated in vivo tumor growth by Notch2 knockdown in triple-negative breast cancer (TNBC) cells. Therapeutic vulnerability of Notch2-altered growth to a small molecule (withaferin A, WA) is also demonstrated. MDA-MB-231 and SUM159 cells were used for the xenograft studies. A variety of technologies were deployed to elucidate the mechanisms underlying tumor growth augmentation by Notch2 knockdown and its reversal by WA, including Fluorescence Molecular Tomography for measurement of tumor angiogenesis in live mice, Seahorse Flux analyzer for ex vivo measurement of tumor metabolism, proteomics, and Luminex-based cytokine profiling. Stable knockdown of Notch2 resulted in accelerated in vivo tumor growth in both cells reflected by tumor volume and/or latency. For example, the wet tumor weight from mice bearing Notch2 knockdown MDA-MB-231 cells was about 7.1-fold higher compared with control (P < 0.0001). Accelerated tumor growth by Notch2 knockdown was highly sensitive to inhibition by a promising steroidal lactone (WA) derived from a medicinal plant. Molecular underpinnings for tumor growth intensification by Notch2 knockdown included compensatory increase in Notch1 activation, increased cellular proliferation and/or angiogenesis, and increased plasma or tumor levels of growth stimulatory cytokines. WA administration reversed many of these effects providing explanation for its remarkable anti-cancer efficacy. Notch2 functions as a tumor growth suppressor in TNBC and WA offers a novel therapeutic strategy for restoring this function.

Proteomic profiling of lymphedema development in mouse model.

The lymphatic vascular system plays an important role in tissue fluid homeostasis. Lymphedema is a chronic, progressive, and incurable condition that leads to lymphatic fluid retention; it may be primary (heritable) or secondary (acquired) in nature. Although there is a growing understanding of lymphedema, methods for the prevention and treatment of lymphedema are still limited. In this study, we investigated differential protein expressions in sham-operated and lymphedema-operated mice for 3 days, using two-dimensional gel electrophoresis (2-DE) and mass spectrometry analysis. Male improved methodology for culturing noninbred (ICR) mice developed lymphedema in the right hindlimb. Twenty functional proteins were found to be differentially expressed between lymphedema induced-right leg tissue and normal left leg tissue. Out of these proteins, the protein levels of apolipoprotein A-1 preprotein, alpha-actinin-3, mCG21744, parkinson disease, serum amyloid P-component precursor, annexin A8, mKIAA0098 protein, and fibrinogen beta chain precursor were differentially upregulated in the lymphedema mice compared with the sham-operated group. Western blotting analysis was used to validate the proteomics results. Our results showing differential up-regulation of serum amyloid P-component precursor, parkinson disease, and apolipoprotein A-1 preprotein in lymphedema model over sham-operated model suggest important insights into pathophysiological target for lymphedema. Copyright © 2016 John Wiley & Sons, Ltd.

Ramie Leaf Extracts Suppresses Adipogenic Differentiation in 3T3-L1 Cells and Pig Preadipocytes.

The present study was carried out to evaluate the anti-obesity effect of different concentrations of extracts of hot air-dried ramie leaf (HR) and freeze-dried ramie leaf (FR) in 3T3-L1 cells and pig preadipocytes. To analyze the effect on cell proliferation, cells were treated with 25 µg/mL or 100 µg/mL HR or FR extract for 2 days. Cell differentiation was evaluated by measuring glycerol-3-phosphate dehydrogenase and lipoprotein lipase (LPL) activities and intracellular triglyceride content. Treatment with either HR or FR extracts inhibited the proliferation of 3T3-L1 cells and pig preadipocytes in a dose-dependent manner. HR extract treatment inhibited the differentiation of both cell types more effectively than FR treatment. The extent of triglyceride accumulation decreased significantly in both cells following either HR or FR treatment. Furthermore, LPL activity significantly decreased after treatment with HR or FR extract. These results indicated that HR and FR extracts may inhibit proliferation and differentiation of 3T3-L1 cells and pig preadipocytes. Further studies are needed to explore the anti-obesity effect of HR and FR extracts.

Growth arrest by the antitumor steroidal lactone withaferin A in human breast cancer cells is associated with down-regulation and covalent binding at cysteine 303 of ß-tubulin.

Withaferin A (WA), a C5,C6-epoxy steroidal lactone derived from a medicinal plant (Withania somnifera), inhibits growth of human breast cancer cells in vitro and in vivo and prevents mammary cancer development in a transgenic mouse model. However, the mechanisms underlying the anticancer effect of WA are not fully understood. Herein, we report that tubulin is a novel target of WA-mediated growth arrest in human breast cancer cells. The G2 and mitotic arrest resulting from WA exposure in MCF-7, SUM159, and SK-BR-3 cells was associated with a marked decrease in protein levels of ß-tubulin. These effects were not observed with the naturally occurring C6,C7-epoxy analogs of WA (withanone and withanolide A). A non-tumorigenic normal mammary epithelial cell line (MCF-10A) was markedly more resistant to mitotic arrest by WA compared with breast cancer cells. Vehicle-treated control cells exhibited a normal bipolar spindle with chromosomes aligned along the metaphase plate. In contrast, WA treatment led to a severe disruption of normal spindle morphology. NMR analyses revealed that the A-ring enone in WA, but not in withanone or withanolide A, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA to Cys(303) of ß-tubulin in MCF-7 cells. Molecular docking indicated that the WA-binding pocket is located on the surface of ß-tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance. These results provide novel insights into the mechanism of growth arrest by WA in breast cancer cells.

Withaferin A inhibits experimental epithelial-mesenchymal transition in MCF-10A cells and suppresses vimentin protein level in vivo in breast tumors.

We have shown previously that withaferin A (WA), a bioactive component of the medicinal plant Withania somnifera, inhibits growth of cultured and xenografted human breast cancer cells and prevents breast cancer development and pulmonary metastasis incidence in a transgenic mouse model. The present study was undertaken to determine if the anticancer effect of WA involved inhibition of epithelial-mesenchymal transition (EMT). Experimental EMT induced by exposure of MCF-10A cells to tumor necrosis factor-α (TNF-α) and transforming growth factor-ß1 (TGF-ß) was partially reversed by treatment with WA but not by its structural analogs withanone or withanolide A. Combined TNF-α and TGF-ß treatments conferred partial protection against MCF-10A cell migration inhibition by WA. Inhibition of TNF-α and TGF-ß-induced MCF-10A cell migration by WA exposure was modestly attenuated by knockdown of E-cadherin protein. MCF-7 and MDA-MB-231 cells exposed to WA exhibited sustained (MCF-7) or transient (MDA-MB-231) induction of E-cadherin protein. On the other hand, the level of vimentin protein was increased markedly after 24 h treatment of MDA-MB-231 cells with WA. WA-induced apoptosis was not affected by vimentin protein knockdown in MDA-MB-231 cells. Protein level of vimentin was significantly lower in the MDA-MB-231 xenografts as well as in MMTV-neu tumors from WA-treated mice compared with controls. The major conclusions of the present study are that (a) WA treatment inhibits experimental EMT in MCF-10A cells, and (b) mammary cancer growth inhibition by WA administration is associated with suppression of vimentin protein expression in vivo.

Differential effects of phenethyl isothiocyanate and D,L-sulforaphane on TLR3 signaling.

Naturally occurring isothiocyanates (ITCs) from cruciferous vegetables are widely studied for their cancer chemopreventive effects. In this study, we investigated the effects of ITCs on TLR signaling, and found that the two most promising ITCs, phenethyl ITCs (PEITC) and D,L-sulforaphane (SFN), have differential effects on dsRNA-mediated innate immune signaling through TLR3. PEITC preferentially inhibited TLR3-mediated IFN regulatory factor 3 (IRF3) signaling and downstream gene expression in vivo and in vitro, whereas SFN caused inhibition of TLR3-mediated NF-κB signaling and downstream gene expression. Mechanistically, PEITC inhibited ligand (dsRNA)-dependent dimerization of TLR3, resulting in inhibition of signaling through IFN regulatory factor 3. In contrast, SFN did not disrupt TLR3 dimerization, indicating that it affects further downstream pathway resulting in NF-κB inhibition. To examine the biological significance of these findings in the context of antitumor activities of these compounds, we used two approaches: first, we showed that dsRNA-mediated apoptosis of tumor cells via TLR3 was inhibited in the presence of PEITC, whereas this response was augmented by SFN treatment; second, in a separate assay measuring anchorage-independent growth and colony formation by immortalized fibroblasts, we made similar observations. Again in this study, PEITC antagonized dsRNA-mediated inhibition of colony formation, whereas SFN enhanced the inhibition. These results indicate biologically relevant functional differences between two structurally similar ITCs and may provide important insights in therapeutic development of these compounds targeted to specific cancer.

Role of mitogen-activated protein kinases and Mcl-1 in apoptosis induction by withaferin A in human breast cancer cells.

Withaferin A (WA), a bioactive constituent of Ayurvedic medicine plant Withania somnifera, is a potent apoptosis inducer in cancer cells but the mechanism of cell death induction is not fully characterized. The present study was undertaken to determine the role of mitogen-activated protein kinases (MAPK), including c-jun NH2 -terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPK, and anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1) in regulation of WA-induced apoptosis using human breast cancer cells. Exposure of MCF-7 (estrogen responsive) and SUM159 (triple negative) human breast cancer cells to WA resulted in increased phosphorylation of ERK, JNK, and p38 MAPK, but these effects were relatively more pronounced in the former cell line than in SUM159. Overexpression of manganese-superoxide dismutase conferred partial protection against WA-mediated hyperphosphorylation of ERK, but not JNK or p38 MAPK. Cell death resulting from WA treatment in MCF-7 cells was significantly augmented by pharmacological inhibition of ERK and p38 MAPK. Interestingly, the WA-induced apoptosis in MCF-7 cells was partially but significantly blocked in the presence of a JNK-specific inhibitor. Pharmacological inhibition of ERK or JNK had no effect on WA-induced apoptosis in SUM159 cells. The WA-treated cells exhibited induction of long and short forms of Mcl-1. RNA interference of Mcl-1 alone triggered apoptosis. Furthermore, the WA-induced cell death in MCF-7 cells was modestly but significantly augmented by knockdown of the Mcl-1 protein. These observations indicate that: MAPK have cell line-specific role in cell death by WA, and Mcl-1 induction confers modest protection against WA-induced apoptosis.

Triphenylphosphonium-functionalized dimeric BODIPY-based nanoparticles for mitochondria-targeting photodynamic therapy.

The dimerization of boron dipyrromethene (BODIPY) moieties is an appealing molecular design approach for developing heavy-atom-free triplet photosensitizers (PSs). However, BODIPY dimer-based PSs generally lack target specificity, which limits their clinical use for photodynamic therapy. This study reports the synthesis of two mitochondria-targeting triphenylphosphonium (TPP)-functionalized meso-ß directly linked BODIPY dimers (BTPP and BeTPP). Both BODIPY dimers exhibited solvent-polarity-dependent singlet oxygen (1O2) quantum yields, with maximum values of 0.84 and 0.55 for BTPP and BeTPP, respectively, in tetrahydrofuran. The compact orthogonal geometry of the BODIPY dimers facilitated the generation of triplet excited states via photoinduced charge separation (CS) and subsequent spin-orbit charge-transfer intersystem crossing (SOCT-ISC) processes and their rates were dependent on the energetic configuration between the frontier molecular orbitals of the two BODIPY subunits. The as-synthesized compounds were amphiphilic and hence formed stable nanoparticles (∼36 nm in diameter) in aqueous solutions, with a zeta potential of ∼33 mV beneficial for mitochondrial targeting. In vitro experiments with MCF-7 and HeLa cancer cells indicated the effective localization of BTPP and BeTPP within cancer-cell mitochondria. Under light irradiation, BTPP and BeTPP exhibited robust photo-induced therapeutic effects in both cell lines, with half-maximal inhibitory concentration (IC50) values of ∼30 and ∼55 nM, respectively.

Critical role for reactive oxygen species in apoptosis induction and cell migration inhibition by diallyl trisulfide, a cancer chemopreventive component of garlic.

Diallyl trisulfide (DATS) is a structurally simple but biologically active constituent of processed garlic with in vivo activity against chemically induced as well as oncogene-driven cancer in experimental rodents. This study offers novel insights into the mechanisms underlying anticancer effects of DATS using human breast cancer cells as a model. Exposure of human breast cancer cells (MCF-7 and MDA-MB-231) and a cell line derived from spontaneously developing mammary tumor of a transgenic mouse (BRI-JM04) to DATS resulted in a dose-dependent inhibition of cell viability that was accompanied by apoptosis induction. A non-tumorigenic normal human mammary cell line (MCF-10A) was resistant to growth inhibition and apoptosis induction by DATS. The DATS-induced apoptosis in MDA-MB-231, MCF-7, and BRI-JM04 cells was associated with reactive oxygen species (ROS) production as evidenced by fluorescence microscopy and flow cytometry using a chemical probe (MitoSOX Red). Overexpression of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) as well as Mn-SOD conferred significant protection against DATS-induced ROS production and apoptotic cell death in MDA-MB-231 and MCF-7 cells. Activation of Bak, but not Bax, resulting from DATS treatment was markedly suppressed by overexpression of Mn-SOD. The DATS treatment caused ROS generation, but not activation of Bax or Bak, in MCF-10A cells. Furthermore, the DATS-mediated inhibition of cell migration was partially but significantly attenuated by Cu,Zn-SOD and Mn-SOD overexpression in association with changes in levels of proteins involved in epithelial-mesenchymal transition. The DATS-mediated induction of heme oxygenase-1 was partially attenuated by overexpression of Mn-SOD. These results provide novel mechanistic insights indicating a critical role for ROS in anticancer effects of DATS.

Saccharified and Fermented Helianthus tuberosus L. Beverage Attenuates High-Fat Diet-Inducible Metabolic Complications in C57BL/6 Mice.

The prevalence of obesity has been recognized as a major public health issue with rapid increase globally. Obesity triggers other metabolic complications, such as diabetes, dyslipidemia, liver diseases, and cardiovascular diseases. Helianthus tuberosus L. (the Jerusalem artichoke) is an important edible plant that may provide health benefits in treating metabolic diseases. In this study, we investigated potential antiobesity effects of saccharified H. tuberosus L. (SH) and its fermented vinegar (fermented H. tuberosus L. [FH]) in a high-fat diet (HFD)-induced obesity murine model. FH exhibited significantly lower pH, Brix, and total sugar content compared with the SH, along with higher radical-scavenging activity. The body weight and adipose tissue weights were significantly decreased with the administration of SH and FH compared with the HFD group. SH and FH groups significantly attenuated hepatomegaly and lipid accumulation. The increased triglyceride (TG) content in obese mice was remarkably lower in the SH and FH groups. SH and FH alleviated serum dyslipidemia and atherogenic risk. Furthermore, expression of adipogenic genes was significantly downregulated after SH and FH supplementation compared with the HFD group. The TG and total cholesterol (TC) content of serum and adipose tissues significantly decreased by SH and FH administration in comparison with the HFD group. Reduced adiposity with SH and FH administration was confirmed by reduced adipocyte size and weight with inhibition of lipoprotein lipase expression. Our study showed that SH and FH, indeed FH was superior to SH, had antiobesity effects by decreasing adiposity, regulating dyslipidemia in systemic tissues, and inhibiting adipogenic gene expression.

Withaferin A causes activation of Notch2 and Notch4 in human breast cancer cells.

Ayurvedic medicine plants continue to draw attention for the discovery of novel anticancer agents. Withaferin A (WA) is one such small-molecule constituent of the ayurvedic medicine plant Withania somnifera with efficacy against cultured and xenografted human breast cancer cells. However, the mechanism underlying anticancer effect of WA is not fully understood. This study was undertaken to determine the role of Notch signaling in anticancer effects of WA using human breast cancer cells as a model. Notably, Notch signaling is often hyperactive in human breast cancers. Exposure of MDA-MB-231 and MCF-7 human breast cancer cells to pharmacological concentrations of WA resulted in cleavage (activation) of Notch2 as well as Notch4, which was accompanied by transcriptional activation of Notch as evidenced by RBP-Jk, HES-1A/B, and HEY-1 luciferase reporter assays. On the other hand, WA treatment caused a decrease in levels of both transmembrane and cleaved Notch1. The WA-mediated activation of Notch was associated with induction of γ-secretase complex components presenilin1 and/or nicastrin. Inhibition of MDA-MB-231 and MDA-MB-468 cell migration resulting from WA exposure was significantly augmented by knockdown of Notch2 as well as Notch4 protein. Activation of Notch2 was not observed in cells treated with withanone or withanolide A, which are structural analogs of WA. The results of this study indicate that WA treatment activates Notch2 and Notch4, which impede inhibitory effect of WA on breast cancer cell migration.

Inhibition of IGF-1 signaling by genistein: modulation of E-cadherin expression and downregulation of ß-catenin signaling in hormone refractory PC-3 prostate cancer cells.

Elevated levels of insulin-like growth factor-1 (IGF-1) are associated with an increased risk of several different cancers, including prostate cancer. Inhibition of IGF-1 and the downstream signaling pathways mediated by the activation of the IGF-1 receptor (IGF-1R) may be involved in inhibiting prostate carcinogenesis. We investigated whether genistein downregulated the IGF-1/IGF-1R signaling pathway and inhibited cell growth in hormone refractory PC-3 prostate cancer cells. Genistein treatment caused a significant inhibition of IGF-1-stimulated cell growth. Flow cytometry analysis revealed that genistein significantly decreased the number of IGF-1-stimulated cells in the G0/G1 phase of the cell cycle. In IGF-1-treated cells, genistein effectively inhibited the phosphorylation of IGF-1R and the phosphorylation of its downstream targets, such as Src, Akt, and glycogen synthase kinase-3ß (GSk-3ß). IGF-1 treatment decreased the levels of E-cadherin but increased the levels of ß-catenin and cyclin D1. However, genistein treatment greatly attenuated IGF-1-induced ß-catenin signaling that correlated with increasing the levels of E-cadherin and decreasing cyclin D1 levels in PC-3 cells. In addition, genistein inhibited T-cell factor/lymphoid enhancer factor (TCF/LEF)-dependent transcriptional activity. These results showed that genistein effectively inhibited cell growth in IGF-1-stimulated PC-3 cells, possibly by inhibiting downstream of IGF-1R activation.

BODIPY nanoparticles functionalized with lactose for cancer-targeted and fluorescence imaging-guided photodynamic therapy.

A series of four lactose-modified BODIPY photosensitizers (PSs) with different substituents (-I, -H, -OCH3, and -NO2) in the para-phenyl moiety attached to the meso-position of the BODIPY core were synthesized; the photophysical properties and photodynamic anticancer activities of these sensitizers were investigated, focusing on the electronic properties of the different substituent groups. Compared to parent BODIPY H, iodine substitution (BODIPY I) enhanced the intersystem crossing (ISC) to produce singlet oxygen (1O2) due to the heavy atom effect, and maintained a high fluorescence quantum yield (ΦF) of 0.45. Substitution with the electron-donating methoxy group (BODIPY OMe) results in a significant perturbation of occupied frontier molecular orbitals and consequently achieves higher 1O2 generation capability with a high ΦF of 0.49, while substitution with the electron-withdrawing nitro group (BODIPY NO2) led a perturbation of unoccupied frontier molecular orbitals and induces a forbidden dark S1 state, which is negative for both fluorescence and 1O2 generation efficiencies. The BODIPY PSs formed water-soluble nanoparticles (NPs) functionalized with lactose as liver cancer-targeting ligands. BODIPY I and OMe NPs showed good fluorescence imaging and PDT activity against various tumor cells (HeLa and Huh-7 cells). Collectively, the BODIPY NPs demonstrated high 1O2 generation capability and ΦF may create a new opportunity to develop useful imaging-guided PDT agents for tumor cells.

Mitochondrion-targeting PEGylated BODIPY dyes for near-infrared cell imaging and photodynamic therapy.

A series of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-based photosensitizers (AmBXI, X = H, M, Br) featuring a cationic mitochondrion-targeting group and near-infrared (NIR) absorption was synthesized. After extending the photosensitizers' π conjugation via Knoevenagel reaction, both the absorbance and emission maxima of AmBXI shifted to the phototherapeutic wavelength range (650-900 nm). Theoretical computations indicate that the introduction of bromine atoms promotes spin-orbit coupling, so that for each additional bromine atom in AmBXI an increase in singlet oxygen quantum yield would be expected (0.3%, 2.2%, and 4.1%, for AmBHI, AmBMI, and AmBBrI, respectively). Moreover, AmBXI photosensitizers exhibited low cytotoxicity in the dark and high phototoxicity, with the half maximal inhibitory concentrations of AmBBrI found to be 46.93 nM and 22.84 nM, while those of AmBMI were 129.7 nM and 58.34 nM in HeLa and MCF-7 cancer cells, respectively. Notably, introduction of a single bromine atom was enough to produce a cytotoxic effect. Furthermore, the presence of a quaternary ammonium group in AmBXI enabled the dyes to localize and stain the negatively charged mitochondria. The results presented herein indicate the straightforward and facile synthesis of NIR-light triggered mitochondrion-targeting photosensitizers.