Potentiation of the effect of erlotinib by genistein in pancreatic cancer: the role of Akt and nuclear factor-kappaB

The epidermal growth factor receptor (EGFR) is a target of new therapies in most nonhematologic cancers. EGFR blockade alone may not be sufficient for the control of growth and invasion of human pancreas cancer because of the independent activation of Akt and nuclear factor-kappaB (NF-kappaB). The expression of EGFR, Akt, and NF-kappaB was determined in six human pancreatic cancer cell lines. Selected cells for specific expression were treated with erlotinib, genistein, gemcitabine, or the combination. Growth inhibition was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and apoptosis was assayed by ELISA. EGFR, phosphorylated EGFR, phosphorylated Akt, and survivin expressions were determined by immunoblotting. Electrophoretic mobility shift assay was used to evaluate the DNA binding activity of NF-kappaB. Genistein significantly increased (P < 0.05) erlotinib-induced growth inhibition and apoptosis in BxPC-3, CAPAN-2, and AsPC-1 cells. In the BxPC-3 cell line, significant down-regulation of EGFR, phosphorylated Akt, NF-kappaB activation, and survivin was observed in the cells treated with the combination compared with the erlotinib-treated cells. In the HPAC and MIAPaCa cell line, no potentiation of the effects of erlotinib by genistein on cell growth or inhibition of the EGFR/Akt/NF-kappaB was observed. Genistein potentiated growth inhibition and apoptosis of the gemcitabine and erlotinib combination in COLO-357 cell line. Genistein potentiates the growth inhibition and apoptosis induced by erlotinib and gemcitabine in certain pancreatic cancer cells. Akt and NF-kappaB inhibition represents one of the mechanisms for the potentiation of erlotinib- and gemcitabine-induced cell death by genistein.     

Role of NF-kappaB and Akt/PI3K in the resistance of pancreatic carcinoma cell lines against gemcitabine-induced cell death

Pancreatic cancer is resistant to almost all cytotoxic drugs. Currently, gemcitabine appears to be the only clinically active drug but, because of pre-existing or acquired chemoresistance of most of the tumor cells, it failed to significantly improve the outcome of pancreatic carcinoma patients. The current study examined the relevance of nuclear factor kappaB (NF-kappaB) and PI3K/Akt in the resistance of five pancreatic carcinoma cell lines towards gemcitabine. Treatment for 24 h with gemcitabine (0.04-20 micro M) led to a strong induction of apoptosis in PT45-P1 and T3M4 cells but not in BxPc-3, Capan-1 and PancTu-1 cells. These resistant cell lines exhibited a high basal NF-kappaB activity in contrast to the sensitive cell lines. Furthermore, gemcitabine showed a dose-dependent induction of NF-kappaB. At a dose of 0.04 micro M, gemcitabine still induced apoptosis in the sensitive cell lines, but did not induce NF-kappaB. In addition, NF-kappaB inhibition by MG132, sulfasalazine or the IkappaBalpha super-repressor strongly diminished the resistance against gemcitabine (0.04-20 micro M). In contrast to this obvious correlation between basal NF-kappaB activity and gemcitabine resistance, PI3K/Akt seems not to be involved in gemcitabine resistance of these cell lines. Neither did the basal Akt activity correlate with the sensitivity towards gemcitabine treatment, nor did the inhibition of PI3K/Akt by LY294002 alter gemcitabine-induced apoptosis. These results indicate that constitutive NF-kappaB activity confers resistance against gemcitabine and that modulation of this activity by pharmacological or genetic approaches may have therapeutical potential when combined with gemcitabine in the treatment of pancreatic carcinoma.     

BRG1 promotes chemoresistance of pancreatic cancer cells through crosstalking with Akt signalling

Gemcitabine is a standard chemotherapeutic agent for locally advanced and metastatic pancreatic cancer. However, the chemoresistance of pancreatic cancer is the major barrier to efficient chemotherapy. Here, we reported that BRG1, a chromatin modulator, was exclusively overexpressed in human pancreatic ductal adenocarcinoma tissues. BRG1 knockdown inhibited PANC-1 and MIA PaCa-2 cell growth in vitro and in vivo, reduced the phosphorylation/activation of Akt and p21^cip/waf, enhanced intrinsic and gemcitabine induced apoptosis and attenuated gemcitabine-induced downregulation of E-cadherin. Moreover, by establishing acquired chemoresistance of MIA PaCa-2 cells in vitro, we found that BRG1 knockdown effectively reversed the chemoresistance to gemcitabine. Surprisingly, inhibiting Akt phosphorylation resulted in BRG1 suppression in pancreatic cancer cells, indicating BRG1 as a new downstream target of Akt signalling. Taken together, our findings suggest that BRG1 promotes both intrinsic and acquired chemoresistance of pancreatic cancer cells, and BRG1 crosstalks with Akt signalling to form a positive feedback loop to promote pancreatic cancer development.     

Wortmannin inhibits pkb/akt phosphorylation and promotes gemcitabine antitumor activity in orthotopic human pancreatic cancer xenografts in immunodeficient mice.

Pancreatic cancer is resistant to almost all classes of cytotoxic agents. Gemcitabine seems to be the current drug of choice. We have recently reported that inhibition of the phosphatidylinositide 3-kinase-protein kinase B (PKB/Akt) cell survival pathway by wortmannin enhances gemcitabine-induced apoptosis in cultured human pancreatic cancer cells (1). The present study investigated the effects of wortmannin on orthotopic human pancreatic cancer xenografts implanted in severe combined immunodeficient mice. Animals were given single i.v. bolus injections of 0.175, 0.35, or 0.7 mg/kg of wortmannin and killed at 0.5, 1, 2, or 4 h after treatment. Phosphorylated PKB/Akt levels in tumor tissues were measured by fluorescence image analysis. Wortmannin was found to inhibit PKB/Akt phosphorylation in a time- and dose-dependent manner, reaching a plateau at 4 h and at 0.7 mg/kg. The levels of phosphorylated PKB/Akt were maximally decreased by approximately 50% relative to the vehicle control. Subsequently, the extent of apoptosis in tumors treated with gemcitabine or wortmannin alone or in combination was determined using terminal deoxynucleotidyl transferase-mediated nick end labeling assay and computerized image analysis. Orthotopic tumors exposed to 80 mg/kg gemcitabine for 48 h and then 0.7 mg/kg wortmannin for 4 h showed a 5-fold increase (P = 0.002) in apoptosis compared with those treated with each agent alone and with the vehicle control. The combination treatment also significantly (P < 0.001) inhibited tumor growth. Taken together, our findings support the potential of phosphatidylinositide 3-kinase inhibitors as adjuncts to conventional chemotherapy in the treatment of pancreatic cancer.     

Zyflamend suppresses growth and sensitizes human pancreatic tumors to gemcitabine in an orthotopic mouse model through modulation of multiple targets

Agents that can potentiate the efficacy of standard chemotherapy against pancreatic cancer are of great interest. Because of their low cost and safety, patients commonly use a variety of dietary supplements, although evidence of their efficacy is often lacking. One such commonly used food supplement is Zyflamend, a polyherbal preparation with potent anti-inflammatory activities and preclinical efficacy against prostate and oral cancer. Whether Zyflamend has any efficacy against human pancreatic cancer alone or in combination with gemcitibine, a commonly used agent, was examined in cell cultures and in an orthotopic mouse model. In vitro, Zyflamend inhibited the proliferation of pancreatic cancer cell lines regardless of p53 status and also enhanced gemcitabine-induced apoptosis. This finding correlated with inhibition of NF-κB activation by Zyflamend and suppression of cyclin D1, c-myc, COX-2, Bcl-2, IAP, survivin, VEGF, ICAM-1 and CXCR4. In nude mice, oral administration of Zyflamend alone significantly inhibited the growth of orthotopically transplanted human pancreatic tumors, and when combined with gemcitabine, further enhanced the antitumor effects. Immunohistochemical and Western blot analyses of tumor tissue showed that the suppression of pancreatic cancer growth correlated with inhibition of proliferation index marker (Ki-67), COX-2, MMP-9, NF-κB and VEGF. Overall, these results suggest that the concentrated multiherb product Zyflamend alone can inhibit the growth of human pancreatic tumors and, in addition, can sensitize pancreatic cancers to gemcitabine through the suppression of multiple targets linked to tumorigenesis.     

RNA interference targeting the M2 subunit of ribonucleotide reductase enhances pancreatic adenocarcinoma chemosensitivity to gemcitabine

Ribonucleotide reductase is emerging as an important determinant of gemcitabine chemoresistance in human cancers. Activity of this enzyme, which catalyses conversion of ribonucleotide 5'-diphosphates to their 2'-deoxynucleotides, is modulated by levels of its M2 subunit (RRM2). Here we show that RRM2 overexpression is associated with gemcitabine chemoresistance in pancreatic adenocarcinoma cells, and that suppression of RRM2 expression using RNA interference mediated by small interfering RNA (siRNA) enhances gemcitabine-induced cytotoxicity in vitro. We demonstrate the ability of systemically administered RRM2 siRNA to suppress tumoral RRM2 expression in an orthotopic xenograft model of pancreatic adenocarcinoma. Synergism between RRM2 siRNA and gemcitabine results in markedly suppressed tumor growth, increased tumor apoptosis and inhibition of metastasis. Our findings confirm the importance of RRM2 in pancreatic adenocarcinoma gemcitabine chemoresistance. This is the first demonstration that systemic delivery of siRNA-based therapy can enhance the efficacy of an anticancer nucleoside analog.     

Usage of the NF-kappaB inhibitor sulfasalazine as sensitizing agent in combined chemotherapy of pancreatic cancer

Sulfasalazine is commonly used as an anti inflammatory agent and is known as a potent inhibitor of NF-kappaB. Some pancreatic carcinomas are characterized by a constitutively elevated NF-kappaB activity accounting for chemoresistance. To elucidate whether blockade of NF-kappaB activity with sulfasalazine is suitable for overcoming this chemoresistance in vivo, we employed a mouse model with subcutaneously xenotransplanted human Capan-1 pancreatic carcinoma cells. Fourteen days upon tumor inoculation, animals were randomized in 6 groups, receiving no treatment, treatment with gemcitabine or with etoposide, either alone or in combination with sulfasalazine, or with sulfasalazine alone. Two therapy regimens were given with a 7-day interval in between. Upon treatment with etoposide or gemcitabine alone, tumor sizes were moderately reduced to 65-68% and 50-65%, respectively, as compared to untreated tumors. Sulfasalazine alone only decreased temporarily the tumor sizes. Sulfasalazine in combination with gemcitabine showed only partially higher reduction in tumor sizes than gemcitabine alone, whereas the combination with etoposide reduced significantly the tumor sizes in all experiments (down to 20%). TUNEL-staining showed higher numbers of apoptotic cells in tumors from the combination groups, in particular with etoposide, and proliferation as indicated by Ki67 staining was strongly reduced. Furthermore, combined treatment of sulfasalazine with the cytostatic drugs led to a decreased blood vessel density. Immunohistochemical staining of the activated p65 subunit showed that sulfasalazine treatment abolished the basal NF-kappaB activity in tumor xenografts. These data imply that the well established anti-inflammatory drug sulfasalazine sensitizes pancreatic carcinoma cells to anti cancer drugs, in particular to etoposide in vivo by inhibition of NF-kappaB. This combined chemotherapy offers great potential for improved anti-tumor responses in pancreatic carcinomas.     

Soy isoflavones enhance radiotherapy in a metastatic prostate cancer model

We previously reported that genistein, the bioactive isoflavone of soybeans, acts as a radiosensitizer for prostate cancer. Pretreatment of tumor cells with genistein potentiated radiation-induced killing in vitro and in orthotopic models in vivo. However, pure genistein promoted increased lymph node metastasis, when administered alone in vivo. We investigated in vitro and in vivo the effects of soy isoflavones (genistein, daidzein and glycitein) as soy pills of similar composition are used in human interventions but not pure genistein. Soy isoflavones inhibited cell survival and potentiated radiation cell killing in PC-3 tumor cells, in vitro. Increased cell killing correlated with inhibition of antiapoptotic molecules Bcl-xL and survivin, upregulation of proapoptotic Bax molecule and PARP cleavage, suggesting activation of apoptotic pathways. In vivo, using the PC-3 orthotopic metastatic mouse model, soy isoflavones and prostate tumor irradiation led to enhanced control of primary tumor growth and metastasis, as observed with pure genistein and radiation. Interestingly, treatment with soy isoflavones did not increase metastasis to para-aortic lymph nodes in contrast to the consistent increase caused by pure genistein. Histologically prostate tumors, treated with soy isoflavones and radiation, showed tumor destruction and in situ tissue alterations, comparable with genistein and radiation effects. However, genistein, but not soy isoflavones, caused induction of HIF1-alpha in prostate tumors, suggesting that induction of hypoxia by pure genistein could contribute to increased metastasis. Our studies demonstrate the safety and potential role of soy isoflavones for enhancing the therapeutic effect of radiotherapy in prostate cancer.     

Resistance of pancreatic cancer to gemcitabine treatment is dependent on mitochondria-mediated apoptosis

Palliative chemotherapy with gemcitabine, a common mode of treatment of pancreatic cancer, has little influence on patients' survival. We investigated the impact of anti-apoptotic Bcl-xL protein and its antagonist Bax on gemcitabine-induced apoptosis in human pancreatic carcinoma cells in vitro and in vivo. The level of Bcl-xL and Bax expression was determined in 3 established pancreatic cancer cell lines that differ in their sensitivity to gemcitabine-mediated apoptosis. Bcl-xL and Bax genes were transduced into Colo357 cells by retroviral infection. In addition, cells were transfected with c-FLIP to assess involvement of CD95 and caspase-8. The impact of Bax/Bcl-xL expression on gemcitabine-sensitivity in vivo was evaluated in orthotopic Colo357 tumors in SCID mice. The apoptotic index revealed a strong inverse correlation between Bcl-xL expression and gemcitabine-induced apoptosis in the pancreatic carcinoma cell lines tested. Caspase-8 and Bid were cleaved in Colo357 cells exposed to gemcitabine, and there was no correlation with either Bcl-xL or with Bax expression. In contrast, the lack of mitochondrial transmembrane potential transition, release of cytochrome-c and absence of caspase-9- and PARP-cleavage showed a strong correlation with Bcl-xL expression. Expression of c-FLIP significantly increased the resistance towards gemcitabine. Orthotopically growing Colo357-bcl-xl tumors in SCID mice were refractory to gemcitabine treatment, and in contrast to the in vitro data, Colo357-bax tumors exhibited a 12-fold greater tumor regression than Colo357-wild-type tumors in the control group. Gemcitabine-induced apoptosis involves the mitochondria-mediated signaling pathway. A functional restoration of this pathway appears to be essential to overcome the resistance mechanisms of pancreatic tumor cells and to improve the response to therapy as demonstrated by Bax overexpression in a clinically relevant tumor model.     

Inactivation of nuclear factor kappaB 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.     

Inactivation of NF-kappaB by genistein is mediated via Akt signaling pathway in breast cancer cells

Genistein, a natural isoflavonoid found in soybean products, has been proposed to be associated with a lower rate of breast cancer in Asian women. Studies from our laboratory and others have shown that genistein can induce apoptosis by regulating the expression of apoptosis-related genes in breast cancer cells. However, the precise molecular mechanism(s) by which genistein induces apoptotic cell death is not clear. In order to investigate such mechanism, we tested the role of Akt and NF-kappaB in genistein-treated MDA-MB-231 breast cancer cells. We found that inhibition of cell growth and induction of apoptosis by genistein are partly mediated through the downregulation of Akt and NF-kappaB pathways. Gel shift assay showed that NF-kappaB DNA-binding activity in MDA-MB-231 cells transfected with Akt cDNA was induced, suggesting that there is a cross-talk between NF-kappaB and Akt signaling pathway. Moreover, we found that genistein could abrogate EGF and Akt induced NF-kappaB activation. From these results, we conclude that the inactivation of NF-kappaB by genistein in MDA-MB-231 breast cancer cells is partly mediated via Akt pathway, which could be useful for rational design of strategies for the prevention and/or treatment of breast cancer.     

RNA interference demonstrates a novel role for integrin-linked kinase as a determinant of pancreatic adenocarcinoma cell gemcitabine chemoresistance.

Integrin-linked kinase (ILK) facilitates signal transduction between extracellular events and important intracellular survival pathways involving protein kinase B/Akt. We examined the role of ILK in determining pancreatic adenocarcinoma cellular chemoresistance to the nucleoside analogue gemcitabine. Cellular ILK expression was quantified by Western blot analysis. We examined the effects of overexpression of active ILK and of ILK knockdown induced by RNA interference on gemcitabine chemoresistance. We also examined the effects of modulating ILK expression on gemcitabine-induced caspase 3-mediated apoptosis, phosphorylation status of Akt (Ser473) and glycogen synthase kinase. Overexpression of ILK increased cellular gemcitabine chemoresistance, whereas ILK knockdown induced chemosensitization via increased caspase 3-mediated apoptosis. ILK knockdown attenuated Akt Ser473 and glycogen synthase kinase phosphorylation, whereas overexpression of constitutively active myristoylated Akt was sufficient to induce significant recovery in gemcitabine chemoresistance in the presence of ILK knockdown. Levels of ILK expression affect gemcitabine chemoresistance in pancreatic adenocarcinoma cells. This novel finding suggests that therapies directed against ILK and its downstream signaling targets may have the potential to enhance the efficacy of gemcitabine-based chemotherapy.     

Selenoprotein P, as a predictor for evaluating gemcitabine resistance in human pancreatic cancer cells

Gemcitabine is a new standard chemotherapeutic agent used in the treatment of pancreatic cancer, but the mechanisms of gemcitabine sensitivity are still controversial. In our study to determine a mechanism that regulates gemcitabine sensitivity, we carried out molecular analysis on the susceptibility of the pancreatic cancer cells. Using a gemcitabine-sensitive pancreatic cancer cell line KLM1, we established a resistant cell line KLM1-R exhibiting a 20-fold IC50-value (the concentration of gemcitabine causing 50% growth inhibition). Microarray analysis of genes showed specific expression of selenoprotein P, one of the anti-oxidants, in the KLM1-R cell line but not in the KLM1 cell line. Administration of selenoprotein P inhibited the gemcitabine-induced cytotoxicity in the pancreatic cell lines. The levels of intracellular reactive oxygen species (ROS) were increased in the KLM1 cells by gemcitabine, but selenoprotein P suppressed the gemcitabine-induced ROS levels. Furthermore interferon-gamma suppressed the expression of selenoprotein P mRNA and increased intracellular ROS level, leading to the recovery of the gemcitabine sensitivity in KLM1-R. These results suggest a novel mechanism that selenoprotein P reduces the intracellular ROS levels, resulting in the insusceptibility to gemcitabine.     

Cisplatin-induced antitumor activity is potentiated by the soy isoflavone genistein in BxPC-3 pancreatic tumor xenografts

BACKGROUND: The activation of nuclear factor kappaB (NF-kappaB) contributes to drug resistance in pancreatic carcinoma. The authors previously showed that the soy isoflavone genistein down-regulates the activation of NF-kappaB in many carcinoma cell lines in vitro. In the current study, they focused their investigation on testing whether the inactivation of NF-kappaB by genistein could enhance cisplatin-induced cell growth inhibition and apoptosis in BxPC-3 cells in vitro and antitumor activity of cisplatin in vivo. METHODS: BxPC-3 cells were preexposed to 25 microM genistein for 24 hours and then exposed to cisplatin (0.5 microM) for an additional 72 hours. A cell growth inhibition assay, an apoptosis assay, and an NF-kappaB electrophoretic mobility shift assay were conducted. For the in vivo study, a xenograft model of BxPC-3 cells in severe combined immunodeficient mice was used. Genistein was given at a dose of 800 microg/kg orally for 5 days, cisplatin was given at a dose of 9 mg/kg as an intraperitoneal bolus, and another group of mice received both cisplatin and genistein (given on Day 1 concurrently followed by genistein for 4 days). RESULTS: The combination of 25 microM genistein with 0.5 microM cisplatin resulted in significantly greater growth inhibition (P < 0.01) and more apoptosis in BxPC-3 cells compared with either agent alone. Preexposure of BxPC-3 cells to genistein abrogated cisplatin-induced activation of NF-kappaB, which appeared to be consistent with the authors' hypothesis. The authors also demonstrated for the first time that the in vivo effect of genistein enhanced the antitumor activity of cisplatin. The tumor weight for the control, genistein, cisplatin, and combined genistein and cisplatin mice was 940 mg, 762 mg, 261 mg, and 108 mg, respectively. Most important, for the first time, the authors observed that the DNA-binding activity of NF-kappaB was inactivated in genistein-treated animal tumors, whereas cisplatin significantly induced NF-kappaB DNA binding activity, and this was completely abrogated in genistein-pretreated tumors that were exposed to cisplatin, consistent with the in vitro data. CONCLUSIONS: Overall, the current results were consistent with the authors' hypothesis and suggested that pretreatment of pancreatic carcinoma cells with genistein down-regulates NF-kappaB activity and contributes toward enhancing the apoptosis-inducing effect of cisplatin, leading to greater antitumor activity in vivo.     

Antitumor and apoptosis-promoting properties of emodin, an anthraquinone derivative from Rheum officinale Baill, against pancreatic cancer in mice via inhibition of Akt activation

Pancreatic adenocarcinoma is one of the most common malignancies worldwide. Gemcitabine is currently the standard first-line chemotherapeutic agent for pancreatic cancer. However, gemcitabine can induce activation of Akt and nuclear factor-κB (NF-κB), which is associated with its chemoresistance. It has been reported that gemcitabine combination therapies result in improved survival outcomes in pancreatic cancer. Therefore, agents that can either enhance the effects of gemcitabine or overcome chemoresistance to the drug are needed for the treatment of pancreatic cancer. Emodin is an active component of Chinese medicinal herbs and can inhibit the activation of Akt and NF-κB. In this study, we investigated whether emodin could enhance the anticancer effect of gemcitabine on pancreatic cancer in vivo. We demonstrated that treatment of gemcitabine combined with emodin efficiently suppressed tumor growth in mice inoculated with pancreatic tumor cells. This treatment paradigm promoted apoptotic cell death and mitochondrial fragmentation. Furthermore, it reduced phosphorylated-Akt (p-Akt) level, NF-κB activation and Bcl-2/Bax ratio, increased caspase-9 and -3 activation, Cytochrome C (CytC) release occurred in combination therapy. Collectively, emodin enhanced the activity of gemcitabine in tumor growth suppression via inhibition of Akt and NF-κB activation, thus promoting the mitochondrial-dependent apoptotic pathway. Therefore, our findings may provide new insights into understanding the pharmacological regulation of emodin on gemcitabine-mediated proapoptosis in pancreatic cancer and may aid in the design of new therapeutic strategies for the intervention of human pancreatic cancers.     

Cancer-associated stromal fibroblasts promote pancreatic tumor progression

Pancreatic adenocarcinoma is characterized by a dense background of tumor associated stroma originating from abundant pancreatic stellate cells. The aim of this study was to determine the effect of human pancreatic stellate cells (HPSC) on pancreatic tumor progression. HPSCs were isolated from resected pancreatic adenocarcinoma samples and immortalized with telomerase and SV40 large T antigen. Effects of HPSC conditioned medium (HPSC-CM) on in vitro proliferation, migration, invasion, soft-agar colony formation, and survival in the presence of gemcitabine or radiation therapy were measured in two pancreatic cancer cell lines. The effects of HPSCs on tumors were examined in an orthotopic murine model of pancreatic cancer by co-injecting them with cancer cells and analyzing growth and metastasis. HPSC-CM dose-dependently increased BxPC3 and Panc1 tumor cell proliferation, migration, invasion, and colony formation. Furthermore, gemcitabine and radiation therapy were less effective in tumor cells treated with HPSC-CM. HPSC-CM activated the mitogen-activated protein kinase and Akt pathways in tumor cells. Co-injection of tumor cells with HPSCs in an orthotopic model resulted in increased primary tumor incidence, size, and metastasis, which corresponded with the proportion of HPSCs. HPSCs produce soluble factors that stimulate signaling pathways related to proliferation and survival of pancreatic cancer cells, and the presence of HPSCs in tumors increases the growth and metastasis of these cells. These data indicate that stellate cells have an important role in supporting and promoting pancreatic cancer. Identification of HPSC-derived factors may lead to novel stroma-targeted therapies for pancreatic cancer.     

Inhibition of nuclear factor kappaB activation in PC3 cells by genistein is mediated via Akt signaling pathway.

Prostate cancer is the second leading cause of cancer-related deaths in menin the United States. Genistein is a prominent isoflavonoid found in soy products and has been proposed to be responsible for lowering the rate of prostate cancer in Asians. However, the molecular mechanism(s) by which genistein elicits its effects on prostate cancer cells has not been fully elucidated. We have previously shown that genistein induces apoptosis and inhibits the activation of nuclear factor kappaB (NF-kappaB) pathway, which could be mediated via Akt signaling pathway, the most important survival pathway in cellular signaling. In this study, we investigated whether there is any cross-talk between Akt and NF-kappaB during genistein-induced apoptosis in PC3 prostate cancer cells. We found that genistein inhibits cell growth and induces apoptotic processes in PC3 prostate cancer cells but not in nontumorigenic CRL-2221 human prostate epithelial cells. Immunoprecipitation, kinase assays, and Western blot analysis showed that genistein specifically inhibits Akt kinase activity and abrogates the epidermal growth factor-induced activation of Akt in prostate cancer cells. NF-kappaB DNA-binding analysis and transfection studies with Akt cDNA and NF-kappaB-Luc constructs revealed that Akt transfection results in the induction of NF-kappaB activation and this is completely inhibited by genistein treatment. Moreover, genistein abrogated the epidermal growth factor-induced activation of NF-kappaB, which was mediated via Akt signaling pathway. From these results, we conclude that the inhibition of Akt and NF-kappaB activity and their cross-talk provide a novel mechanism by which genistein inhibits cell growth and induces apoptotic processes in tumorigenic but not in nontumorigenic prostate epithelial cells.