Kaoru Kiguchi

In vitro and in vivo anti-tumor activities of a gemcitabine derivative carried by nanoparticles

Gemcitabine (Gemzar(®)) is the first line treatment for pancreatic cancer and often used in combination therapy for non-small cell lung, ovarian, and metastatic breast cancers. Although extremely toxic to a variety of tumor cells in culture, the clinical outcome of gemcitabine treatment still needs improvement. In the present study, a new gemcitabine nanoparticle formulation was developed by incorporating a previously reported stearic acid amide derivative of gemcitabine into nanoparticles prepared from lecithin/glyceryl monostearate-in-water emulsions. The stearoyl gemcitabine nanoparticles were cytotoxic to tumor cells in culture, although it took a longer time for the gemcitabine in the nanoparticles to kill tumor cells than for free gemcitabine. In mice with pre-established model mouse or human tumors, the stearoyl gemcitabine nanoparticles were significantly more effective than free gemcitabine in controlling the tumor growth. PEGylation of the gemcitabine nanoparticles with polyethylene glycol (2000) prolonged the circulation of the nanoparticles in blood and increased the accumulation of the nanoparticles in tumor tissues (>6-fold), but the PEGylated and un-PEGylated gemcitabine nanoparticles showed similar anti-tumor activity in mice. Nevertheless, the nanoparticle formulation was critical for the stearoyl gemcitabine to show a strong anti-tumor activity. It is concluded that for the gemcitabine derivate-containing nanoparticles, cytotoxicity data in culture may not be used to predict their in vivo anti-tumor activity, and this novel gemcitabine nanoparticle formulation has the potential to improve the clinical outcome of gemcitabine treatment.

EGFR-targeted stearoyl gemcitabine nanoparticles show enhanced anti-tumor activity

Previously, it was shown that a novel 4-(N)-stearoyl gemcitabine nanoparticle formulation was more effective than gemcitabine hydrochloride in controlling the growth of model mouse or human tumors pre-established in mice. In the present study, the feasibility of targeting the stearoyl gemcitabine nanoparticles (GemC18-NPs) into tumor cells that over-express epidermal growth factor receptor (EGFR) to more effectively control tumor growth was evaluated. EGFR is over-expressed in a variety of tumor cells, and EGF is a known natural ligand of EGFR. Recombinant murine EGF was conjugated onto the GemC18-NPs. The ability of the EGF to target the GemC18-NPs to human breast adenocarcinoma cells that expressed different levels of EGFR was evaluated in vitro and in vivo. In culture, the extent to which the EGF-conjugated GemC18-NPs were taken up by tumor cells was correlated to the EGFR density on the tumor cells, whereas the uptake of untargeted GemC18-NPs exhibited no difference among those same cell lines. The relative cytotoxicity of the EGF-conjugated GemC18-NPs to tumor cells in culture was correlated to EGFR expression as well. In vivo, EGFR-over-expressing MDA-MB-468 tumors in mice treated with the EGF-conjugated GemC18-NPs grew significantly slower than in mice treated with untargeted GemC18-NPs, likely due to that the EGF-GemC18-NPs were more anti-proliferative, anti-angiogenic, and pro-apoptotic. Fluorescence intensity data from ex vivo imaging showed that the EGF on the nanoparticles helped increase the accumulation of the GemC18-NPs into MDA-MB-468 tumors pre-established in mice by more than 2-fold as compared to the un-targeted GemC18-NPs. In conclusion, active targeting of the GemC18-NPs into EGFR-over-expressed tumors can further enhance their anti-tumor activity.

Stat3 Binds to mtDNA and Regulates Mitochondrial Gene Expression in Keratinocytes

The nuclear transcription factor Stat3 has recently been reported to have a localized mitochondrial regulatory function. Current data suggest that mitochondrial Stat3 (mitoStat3) is necessary for maximal mitochondrial activity and for Ras-mediated transformation independent of Stat3 nuclear activity. We have previously shown that Stat3 plays a pivotal role in epithelial carcinogenesis. Therefore, the aim of the current study was to determine the role of mitoStat3 in epidermal keratinocytes. Herein, we show that normal and neoplastic keratinocytes contain a pool of mitoStat3. EGF and TPA induce Stat3 mitochondrial translocation mediated through phosphorylation of Stat3 at Ser727. In addition, we report that mitoStat3 binds mitochondrial DNA (mtDNA) and associates with the mitochondrial transcription factor TFAM. Furthermore, Stat3 ablation resulted in an increase of mitochondrial encoded gene transcripts. An increase in key nuclear-encoded metabolic genes, PGC-1α and NRF-1, was also observed in Stat3 null keratinocytes, however no changes in nuclear-encoded ETC gene transcripts or mtDNA copy number were observed. Collectively, our findings suggest a heretofore-unreported function for mitoStat3 as a potential mitochondrial transcription factor in keratinocytes. This mitoStat3-mtDNA interaction may represent an alternate signaling pathway that could alter mitochondrial function and biogenesis and play a role in tumorigenesis.

Silencing of ribonucleotide reductase subunit M1 potentiates the antitumor activity of gemcitabine in resistant cancer cells

Gemcitabine is a deoxycytidine analog used for the treatment of a wide range of solid tumors. Its efficacy is however often reduced due to the development of resistance. Ribonucleotide reductase M1 subunit (RRM1) is a key determinant of gemcitabine resistance, and tumor cells that overexpress RRM1 are resistant to the cytotoxicity of gemcitabine. In the present study, we showed that RRM1-specific small interfering RNA (siRNA), when complexed with polyethylenimine, effectively downregulated the expression of RRM1 protein in mouse tumor cells that overexpress RRM1, both in vitro and in vivo. More importantly, systemic administration of the RRM1-specific siRNA significantly inhibited the growth of RRM1-overexpressing tumors in mice and sensitized the tumors to gemcitabine treatment. These findings suggest that silencing RRM1 expression using siRNA could potentially be an effective strategy to overcome gemcitabine resistance.

Proteomic and pathway analyses reveal a network of inflammatory genes associated with differences in skin tumor promotion susceptibility in DBA/2 and C57BL/6 mice

Genetic susceptibility to two-stage skin carcinogenesis is known to vary significantly among different stocks and strains of mice. In an effort to identify specific protein changes or altered signaling pathways associated with skin tumor promotion susceptibility, a proteomic approach was used to examine and identify proteins that were differentially expressed in epidermis between promotion-sensitive DBA/2 and promotion-resistant C57BL/6 mice following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). We identified 19 differentially expressed proteins of which 5 were the calcium-binding proteins annexin A1, parvalbumin α, S100A8, S100A9, and S100A11. Further analyses revealed that S100A8 and S100A9 protein levels were also similarly differentially upregulated in epidermis of DBA/2 versus C57BL/6 mice following topical treatment with two other skin tumor promoters, okadaic acid and chrysarobin. Pathway analysis of all 19 identified proteins from the present study suggested that these proteins were components of several networks that included inflammation-associated proteins known to be involved in skin tumor promotion (e.g. TNF-α, NFκB). Follow-up studies revealed that Tnf, Nfkb1, Il22, Il1b, Cxcl1, Cxcl2 and Cxcl5 mRNAs were highly expressed in epidermis of DBA/2 compared with C57BL/6 mice at 24h following treatment with TPA. Furthermore, NFκB (p65) was also highly activated at the same time point (as measured by phosphorylation at ser276) in epidermis of DBA/2 mice compared with C57BL/6 mice. Taken together, the present data suggest that differential expression of genes involved in inflammatory pathways in epidermis may play a key role in genetic differences in susceptibility to skin tumor promotion in DBA/2 and C57BL/6 mice.

Constitutive Stat3 activation alters behavior of hair follicle stem and progenitor cell populations

STATs play crucial roles in a wide variety of biological functions, including development, proliferation, differentiation and migration as well as in cancer development. In the present study, we examined the impact of constitutive activation of Stat3 on behavior of keratinocytes, including keratinocyte stem cells (KSC) in vivo. BK5.Stat3C transgenic (Tg) mice, which express a constitutively active form of Stat3 (Stat3C) in the basal layer of the epidermis and in the bulge region KSCs exhibited a significantly reduced number of CD34+/α6 integrin+ cells compared to non‐transgenic (NTg) littermates. There was a concomitant increase in the Lgr‐6, Lrig‐1, and Sca‐1 populations in the Tg mice in contrast to the CD34 and Keratin‐15 positive population. In addition, increased expression of c‐myc, β‐catenin, and epithelial–mesenchymal transition (EMT)‐related genes as well as decreased expression of α6‐integrin was observed in the hair follicles of Tg mice. Notably, Sca‐1 was found to be a direct transcriptional target of Stat3 in keratinocytes. The current data suggest that elevated Stat3 activity leads to depletion of hair follicle KSCs along with a concomitant increase of stem/progenitor cells above the bulge region. Overall, the current data indicate that Stat3 plays an important role in keratinocyte stem/progenitor cell homeostasis.

Transcutaneous DNA immunization following waxing-based hair depilation

Transcutaneous DNA immunization is an attractive immunization approach. Previously, we reported that transcutaneous immunization by applying plasmid DNA onto a skin area wherein the hair follicles had been induced into growth stage by cold waxing-based hair plucking significantly enhanced the resultant immune responses. In the present study, using a plasmid that encodes the Bacillus anthracis protective antigen (PA63) gene fragment, it was shown that the anti-PA63 antibody responses induced by applying the plasmid onto a skin area where the hair was plucked by warm waxing were significantly stronger than by cold waxing, very likely because the warm waxing-based hair depilation significantly i) enhanced the uptake (or retention) of the plasmid in the application area and ii) enhanced the expression of the transfected gene in the follicular and interfollicular epidermis in the skin. The antibody response induced by transcutaneous DNA immunization was hair cycle dependent, because the plasmid needed to be applied within 5days after the hair plucking to induce a strong antibody response. The antibody responses were not affected by whether the expressed PA63 protein, as an antigen, was secreted or cell surface bound. Finally, this strategy of enhancing the immune responses induced by transcutaneous DNA immunization following warm waxing-based hair depilation was not limited to the PA63 as an antigen, because immunization with a plasmid that encodes the HIV-1 env gp160 gene induced a strong anti-gp160 response as well. Transcutaneous DNA immunization by modifying the hair follicle cycle may hold a great promise in inducing strong and functional immune responses.

Selective Reduction in α‐Hydroxypalmitic Acid‐containing Sphingomyelin and Concurrent Increase in Hydroxylated Ceramides in Murine Skin Tumors Induced by an Initiation‐promotion Regimen

The sphingomyelin cycle is activated to accumulate ceramides in the process of epidermal differentiation. We found that sphingomyelin in the epidermis of 4 different murine strains gave three bands on TLC, the lower band containing α‐hydroxypalmitic acid (C16h:0(α)). However, in the papillomas induced in the skin of SENCAR and SSIN mice by initiation with 7,12‐dimethylbenz[α] anthracene followed by promotion with 12‐O‐tetradecanoylphorbol acetate, the concentration of C16h:0(α)‐ containing sphingomyelin was selectively diminished with a concomitant increase in the concentrations of the ceramides containing α‐hydroxy fatty acids. These findings indicate a possible involvement of the selective hydrolysis of α‐hydroxy fatty acid‐containing sphingomyelin in the process of tumorigenesis in mouse skin.

Bile acid accelerates erbB2-induced pro-tumorigenic activities in biliary tract cancer

Although very few studies have addressed the molecular and cellular mechanisms underlying the development of biliary tract cancer (BTC), several lines of evidence suggest a role for the erbB receptor family. Overexpression and activation of erbB2 has been reported in a significant percentage of human BTC. Further, we previously reported that overexpression of erbB2 basal epithelial cells of the biliary tract (BK5.erbB2 mouse) led to the development of BTC. However, the mechanisms by which erbB2 overexpression led to the spontaneous development of tumors specifically in the biliary tract are not completely understood. The goals of the current study were to (1) determine whether a cooperative relationship between bile acid exposure and erbB2 activation exists during biliary tract carcinogenesis and (2) to characterize the mechanism(s) underlying bile acid‐mediated biliary tract carcinogenesis in cells with activated erbB2. In this study, we demonstrated that the secondary conjugated bile acid, taurochenodeoxycholic acid (TCDC), increased proliferation of primary cultured gallbladder epithelial cells from BK5.erbB2 mice and human BTC cells. TCDC treatment activated EGFR/erbB2 and downstream signaling molecules in both primary cultured cells and human BTC cells. TCDC also increased the expression of epidermal growth factor receptor (EGFR) ligands and TACE activity in human BTC cells. Inhibition of src activation led to attenuation of bile‐induced upregulation of TACE activity as well as signaling through the EGFR/erbB2, suggesting that during the development of BTC erbB2 overexpression/activation accelerates the bile acid‐induced signaling cascade: bile acidu2009→u2009srcu2009→u2009TACEu2009→u2009EGFR/erbB2u2009→u2009downstream signaling. We also provide direct evidence that bile acids possess tumor promoting capacity in epithelial cells overexpressing erbB2 using the two‐stage skin carcinogenesis model. Collectively these findings suggest cooperative roles for bile acid and erbB2 activation in epithelial cell proliferation; bile acid appears to accelerate erbB2‐induced pro‐tumorigenic activities in the biliary tract and skin.

Impact of mTORC1 inhibition on keratinocyte proliferation during skin tumor promotion in wild-type and BK5.AktWT mice.

In this study, we examined the impact of rapamycin on mTORC1 signaling during 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced keratinocyte proliferation and skin tumor promotion in both wild‐type (FVB/N) and BK5.AktWT mice. TPA activated mTORC1 signaling in a time‐dependent manner in cultured primary mouse keratinocytes and a mouse keratinocyte cell line. Early activation (15–30u2009min) of mTORC1 signaling induced by TPA was mediated in part by PKC activation, whereas later activation (2–4u2009h) was mediated by activation of EGFR and Akt. BK5.AktWT transgenic mice, where Akt1 is overexpressed in basal epidermis, are highly sensitive to TPA‐induced epidermal proliferation and two‐stage skin carcinogenesis. Targeting mTORC1 with rapamycin effectively inhibited TPA‐induced epidermal hyperplasia and hyperproliferation as well as tumor promotion in a dose‐dependent manner in both wild‐type and BK5.AktWT mice. A significant expansion (∼threefold) of the label retaining cell (LRC) population per hair follicle was observed in BK5.AktWT mice compared to FVB/N mice. There was also a significant increase in K15 expressing cells in the hair follicle of transgenic mice that coincided with expression of phospho‐Akt, phospho‐S6K, and phospho‐PRAS40, suggesting an important role of mTORC1 signaling in bulge‐region keratinocyte stem cell (KSC) homeostasis. After 2 weeks of TPA treatment, LRCs had moved upward into the interfollicular epidermis from the bulge region of both wild‐type and BK5.AktWT mice. TPA‐mediated LRC proliferation and migration was significantly inhibited by rapamycin. Collectively, the current data indicate that signaling through mTORC1 contributes significantly to the process of skin tumor promotion through effects on proliferation of the target cells for tumor development.