Synergistic effect of therapeutic stem cells expressing cytosine deaminase and interferon-beta via apoptotic pathway in the metastatic mouse model of breast cancer.

As an approach to improve treatment of breast cancer metastasis to the brain, we employed genetically engineered stem cells (GESTECs, HB1.F3 cells) consisting of neural stem cells (NSCs) expressing cytosine deaminase and the interferon-beta genes, HB1.F3.CD and HB1.F3.CD.IFN-ß. In this model, MDA-MB-231/Luc breast cancer cells were implanted in the right hemisphere of the mouse brain, while pre-stained GESTECs with redfluorescence were implanted in the contralateral brain. Two days after stem cells injection, 5-fluorocytosine (5-FC) was administrated via intraperitoneal injection. Histological analysis of extracted brain confirmed the therapeutic efficacy of GESTECs in the presence of 5-FC based on reductions in density and aggressive tendency of breast cancer cells, as well as pyknosis, karyorrhexis, and karyolysis relative to a negative control. Additionally, expression of PCNA decreased in the stem cells treated group. Treatment of breast cancer cells with 5-fluorouracil (5-FU) increased the expression of pro-apoptotic and anti-proliferative factor, BAX and p21 protein through phosphorylation of p53 and p38. Moreover, analysis of stem cell migratory ability revealed that MDA-MB-231 cells endogenously secreted VEGF, and stem cells expressed their receptor (VEGFR2). To confirm the role of VEGF/VEGFR2 signaling in tumor tropism of stem cells, samples were treated with the VEGFR2 inhibitor, KRN633. The number of migrated stem cells decreased significantly in response to KRN633 due to Erk1/2 activation and PI3K/Akt inhibition. Taken together, these results indicate that treatment with GESTECs, particularly HB1.F3.CD.IFN-ß co-expressing CD.IFN-ß, may be a useful strategy for treating breast cancer metastasis to the brain in the presence of a prodrug.

Minimal Pleural Effusion in Small Cell Lung Cancer: Proportion, Mechanisms, and Prognostic Effect.

PURPOSE: To determine the frequency and investigate possible mechanisms and prognostic relevance of minimal (<10-mm thickness) pleural effusion in patients with small cell lung cancer (SCLC). MATERIALS AND METHODS: The single-center retrospective study was approved by the institutional review board of the hospital, and informed consent was waived by the patients. A cohort of 360 consecutive patients diagnosed with SCLC by using histologic analysis was enrolled in this study. Based on the status of pleural effusion on chest computed tomographic (CT) scans at diagnosis, patients were classified into three groups: no pleural effusion, minimal pleural effusion, and malignant pleural effusion. Eighteen variables related to patient, environment, stage, and treatment were included in the final model as potential confounders. RESULTS: Minimal pleural effusion was present in 74 patients (20.6%) and malignant pleural effusion in 83 patients (23.0%). Median survival was significantly different in patients with no, minimal, or malignant pleural effusion (median survival, 11.2, 5.93, and 4.83 months, respectively; P < .001, log-rank test). In the fully adjusted final model, patients with minimal pleural effusion had a significantly increased risk of death compared with those with no pleural effusion (adjusted hazard ratio, 1.454 [95% confidence interval: 1.012, 2.090]; P = .001). The prognostic effect was significant in patients with stage I-III disease (adjusted hazard ratio, 2.751 [95% confidence interval: 1.586, 4.773]; P < .001), but it disappeared in stage IV disease. An indirect mechanism representing mediastinal lymphadenopathy was responsible for the accumulation in all but one patient with minimal pleural effusion. CONCLUSIONS: Minimal pleural effusion is a common clinical finding in staging SCLC. Its presence is associated with worse survival in patients and should be considered when CT scans are interpreted.

Additional effects of engineered stem cells expressing a therapeutic gene and interferon-ß in a xenograft mouse model of endometrial cancer.

Endometrial cancer is the most common gynecologic malignancy in women worldwide. In the present study, we evaluated the effects of neural stem cell-directed enzyme/prodrug therapy (NDEPT) designed to more selectively target endometrial cancer. For this, we employed two different types of neural stem cells (NSCs), HB1.F3.CD and HB1.F3.CD.IFN-ß cells. Cytosine deaminase (CD) can convert the non-toxic prodrug, 5-fluorocytosine (5-FC), into a toxic agent, 5-fluorouracil (5-FU), which inhibits DNA synthesis. IFN-ß is a powerful cytotoxic cytokine that is released by activated immune cells or lymphocytes. In an animal model xenografted with endometrial Ishikawa cancer cells, the stem cells stained with CM-DiI were injected into nearby tumor masses and 5-FC was delivered by intraperitoneal injection. Co-expression of CD and IFN-ß significantly inhibited the growth of cancer (~50-60%) in the presence of 5-FC. Among migration-induced factors, VEGF gene was highly expressed in endometrial cancer cells. Histological analysis showed that the aggressive nature of cancer was inhibited by 5-FC in the mice treated with the therapeutic stem cells. Furthermore, PCNA expression was more decreased in HB1.F3.CD.IFN-ß treated mice rather than HB1.F3.CD treated mice. To confirm the in vitro combined effects of 5-FU and IFN-ß, 5-FU was treated in Ishikawa cells. 5-FU increased the IFN-ß/receptor 2 (IFNAR2) and BXA levels, indicating that 5-FU increased sensitivity of endometrial cancer cells to IFN-ß, leading to apoptosis of cancer cells. Taken together, these results provide evidence for the efficacy of therapeutic stem cell-based immune therapy involving the targeted expression of CD and IFN-ß genes at endometrial cancer sites.

Interpretation of a heterogeneous radiological response as tumor heterogeneity or a non-tumor diagnosis: A case report.

Heterogeneous radiological responses (HRRs) among tumor lesions are usually observed following chemotherapy or radiation treatment in cancer patients. When HRR is observed after chemotherapy or radiation treatment, a change in anticancer treatment is recommended due to the clinically high suspicion of resistance in the majority of cases. The present study reports the case report of a patient with limited-stage small cell lung cancer, diagnosed by bronchoscopy, who received concurrent chemoradiation therapy. Upon response evaluation, the majority of lesions irradiated had nearly completely disappeared following treatment, but one lesion had apparently increased in size. For histological confirmation, a percutaneous needle biopsy for the lesion was performed, however, non-specific necrosis was found and the results were inconclusive for the differentiation of other causes from tumor necrosis. Several acid-fast bacilli were identified on Ziehl-Neelsen staining for the differential diagnosis. This case suggests that a non-tumor diagnosis should be considered when HRR presents after treatment that is expected to result in a higher response rate, particularly in tuberculosis endemic areas.

Alteration of epithelial-mesenchymal transition markers in human normal ovaries and neoplastic ovarian cancers.

Most ovarian cancers originate in the ovarian surface epithelium (OSE). Ovarian cancers might undergo epithelial-mesenchymal transition (EMT) in response to various mediators or regulators such as EMT-inducing factors. In this study, ovarian tumor specimens from patients were analyzed to demonstrate alteration of EMT-related markers according to benign and malignant types of ovarian cancers. In the three ovarian cancer cell lines, OVCAR-3, SKOV-3, and BG-1, the expression of epithelial (E-cadherin) and mesenchymal (vimentin) cell markers was identified by RNA and protein analysis. OVCAR-3 and BG-1 cells strongly expressed E-cadherin as well as morphological features such as epithelial cells, but vimentin was not observed. In contrast to these cancer cells, SKOV-3 showed a phenotype typical of mesenchymal cells. Alteration of EMT markers and EMT-related transcriptional factors were confirmed in clinical ovarian tissue samples obtained from 74 patients. E-cadherin was expressed in 57.1% of benign tumors, while vimentin was expressed in 83.3% of normal ovaries by immunohistochemistry (IHC) analysis of E-cadherin and vimentin revealed the phenomenon in the tissue specimens. Evaluation of the EMT-associated transcriptional factors Snail, Slug, and Twist revealed that Snail was overexpressed by 7.1-fold in malignant ovarian cancer compared to normal ovaries or benign tumors. Although expression levels of other factors were higher in benign and malignant ovarian tumors, they were not closely correlated with the aforementioned ovarian cancer types. Overall, Snail may affect the EMT process in ovarian cancer development and upregulation of Snail expression followed by the downregulation of E-cadherin enhances the invasiveness of ovarian cancer.

Co-treatment with therapeutic neural stem cells expressing carboxyl esterase and CPT-11 inhibit growth of primary and metastatic lung cancers in mice.

In this study, neural stem cells (NSCs)-derived enzyme/prodrug therapy (NDEPT) was used to treat primary lung cancer or metastatic lung cancer in the brain. To confirm the anti-tumor effect of NSCs expressing carboxyl esterase (CE), A549 lung cancer cells were treated with HB1.F3.CE cells and CPT-11. A significant decrease in the viability/proliferation of lung cancer cells was observed compared to negative controls or cells treated with CPT-11 alone. To produce a mouse model of primary lung cancer or lung cancer metastasis to the brain, A549 cells were implanted in the dorsal area of the mouse or right hemisphere. CM-DiI pre-stained stem cells were implanted near the primary lung cancer tumor mass or in the contralateral brain. Two days after stem cells injection, mice were inoculated with CPT-11 (13.5 kg/mouse/day) via intraperitoneal injection. In the primary lung cancer mouse models, tumor mass was 80% lower in response to HB1.F3.CE in conjunction with CPT-11, while it was only reduced by 40% in the group treated with CPT-11 alone. Additionally, therapeutic efficacy of co-treatment with stem cells and CPT-11 was confirmed by detection of apoptosis and necrosis in primary and metastatic lung cancer tissues. By secreting VEGF, tumor cells modulate Erk1/2 and Akt signaling and migration of stem cells. This further increased tumor-selectivity of stem cell/prodrug co-therapy. Overall, these results indicate that NSCs expressing the therapeutic gene may be a powerful tool for treatment of primary lung cancer or metastasis of lung cancer to the brain.

Role of the epithelial-mesenchymal transition and its effects on embryonic stem cells.

The epithelial-mesenchymal transition (EMT) is important for embryonic development and the formation of various tissues or organs. However, EMT dysfunction in normal cells leads to diseases, such as cancer or fibrosis. During the EMT, epithelial cells are converted into more invasive and active mesenchymal cells. E-box-binding proteins, including Snail, ZEB and helix-loop-helix family members, serve as EMT-activating transcription factors. These transcription factors repress the expression of epithelial markers, for example, E-cadherin, rearrange the cytoskeleton and promote the expression of mesenchymal markers, such as vimentin, fibronectin and other EMT-activating transcription factors. Signaling pathways that induce EMT, including transforming growth factor-ß, Wnt/glycogen synthase kinase-3ß, Notch and receptor tyrosine kinase signaling pathways, interact with each other for the regulation of this process. Although the mechanism(s) underlying EMT in cancer or embryonic development have been identified, the mechanism(s) in embryonic stem cells (ESCs) remain unclear. In this review, we describe the underlying mechanisms of important EMT factors, indicating a precise role for EMT in ESCs, and characterize the relationship between EMT and ESCs.

Methoxychlor and triclosan stimulates ovarian cancer growth by regulating cell cycle- and apoptosis-related genes via an estrogen receptor-dependent pathway.

Methoxychlor and triclosan are emergent or suspected endocrine-disrupting chemicals (EDCs). Methoxychlor [MXC; 1,1,1-trichlor-2,2-bis (4-methoxyphenyl) ethane] is an organochlorine pesticide that has been primarily used since dichlorodiphenyltrichloroethane (DDT) was banned. In addition, triclosan (TCS) is used as a common component of soaps, deodorants, toothpastes, and other hygiene products at concentrations up to 0.3%. In the present study, the potential impact of MXC and TCS on ovarian cancer cell growth and underlying mechanism(s) was examined following their treatments in BG-1 ovarian cancer cells. As results, MXC and TCS induced BG-1 cell growth via regulating cyclin D1, p21 and Bax genes related with cell cycle and apoptosis. A methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay confirmed that the proliferation of BG-1 ovarian cancer cells was stimulated by MXC (10(-6), 10(-7), 10(-8), and 10(-9)M) or TCS (10(-6), 10(-7), 10(-8), and 10(-9)M). Treatment of BG-1 cells with MXC or TCS resulted in the upregulation of cyclin D1 and downregulation of p21 and Bax transcriptions. In addition, the protein level of cyclin D1 was increased by MXC or TCS while p21 and Bax protein levels appeared to be reduced in these cells. Furthermore, MXC- or TCS-induced alterations of these genes were reversed in the presence of ICI 182,780 (10(-7)M), suggesting that the changes in these gene expressions may be regulated by an ER-dependent signaling pathway. In conclusion, the results of our investigation indicate that two potential EDCs, MXC and TCS, may stimulate ovarian cancer growth by regulating cell cycle- and apoptosis-related genes via an ER-dependent pathway.

Selective antitumor effect of neural stem cells expressing cytosine deaminase and interferon-beta against ductal breast cancer cells in cellular and xenograft models.

Due to their inherent tumor-tropic properties, genetically engineered stem cells may be advantageous for gene therapy treatment of various human cancers, including brain, liver, ovarian, and prostate malignancies. In this study, we employed human neural stem cells (HB1.F3; hNSCs) transduced with genes expressing Escherichia coli cytosine deaminase (HB1.F3.CD) and human interferon-beta (HB1.F3.CD.IFN-ß) as a treatment strategy for ductal breast cancer. CD can convert the prodrug 5-fluorocytosine (5-FC) to its active chemotherapeutic form, 5-fluorouracil (5-FU), which induces a tumor-killing effect through DNA synthesis inhibition. IFN-ß also strongly inhibits tumor growth by the apoptotic process. RT-PCR confirmed that HB1.F3.CD cells expressed CD and HB1.F3.CD.IFN-ß cells expressed both CD and IFN-ß. A modified transwell migration assay showed that HB1.F3.CD and HB1.F3.CD.IFN-ß cells selectively migrated toward MCF-7 and MDA-MB-231 human breast cancer cells. In hNSC-breast cancer co-cultures the viability of breast cancer cells which were significantly reduced by HB1.F3.CD or HB1.F3.CD.IFN-ß cells in the presence of 5-FC. The tumor inhibitory effect was greater with the HB1.F3.CD.IFN-ß cells, indicating an additional effect of IFN-ß to 5-FU. In addition, the tumor-tropic properties of these hNSCs were found to be attributed to chemoattractant molecules secreted by breast cancer cells, including stem cell factor (SCF), c-kit, vascular endothelial growth factor (VEGF), and VEGF receptor 2. An in vivo assay performed using MDA-MB-231/luc breast cancer mammary fat pad xenografts in immunodeficient mice resulted in 50% reduced tumor growth and increased long-term survival in HB1.F3.CD and HB1.F3.CD.IFN-ß plus 5-FC treated mice relative to controls. Our results suggest that hNSCs genetically modified to express CD and/or IFN-ß genes can be used as a novel targeted cancer gene therapy.

Development and application of neural stem cells for treating various human neurological diseases in animal models.

Stem cells derived from adult tissues or the inner cell mass (ICM) of embryos in the mammalian blastocyst (BL) stage are capable of self-renewal and have remarkable potential for undergoing lineage-specific differentiation under in vitro culturing conditions. In particular, neural stem cells (NSCs) that self-renew and differentiate into major cell types of the brain exist in the developing and adult central nervous system (CNS). The exact function and distribution of NSCs has been assessed, and they represent an interesting population that includes astrocytes, oligodendrocytes, and neurons. Many researchers have demonstrated functional recovery in animal models of various neurological diseases such as stroke, Parkinson's disease (PD), brain tumors, and metastatic tumors. The safety and efficacy of stem cell-based therapies (SCTs) are also being evaluated in humans. The therapeutic efficacy of NSCs has been shown in the brain disorder-induced animal models, and animal models may be well established to perform the test before clinical stage. Taken together, data from the literature have indicated that therapeutic NSCs may be useful for selectively treating diverse types of human brain diseases without incurring adverse effects.

Anticancer effects of the engineered stem cells transduced with therapeutic genes via a selective tumor tropism caused by vascular endothelial growth factor toward HeLa cervical cancer cells.

The aim of the present study was to investigate the therapeutic efficacy of genetically engineered stem cells (GESTECs) expressing bacterial cytosine deaminase (CD) and/or human interferon-beta (IFN-ß) gene against HeLa cervical cancer and the migration factors of the GESTECs toward the cancer cells. Anticancer effect of GESTECs was examined in a co-culture with HeLa cells using MTT assay to measure cell viability. A transwell migration assay was performed so as to assess the migration capability of the stem cells to cervical cancer cells. Next, several chemoattractant ligands and their receptors related to a selective migration of the stem cells toward HeLa cells were determined by real-time PCR. The cell viability of HeLa cells was decreased in response to 5-fluorocytosine (5-FC), a prodrug, indicating that 5-fluorouracil (5-FU), a toxic metabolite, was converted from 5-FC by CD gene and it caused the cell death in a co-culture system. When IFN-ß was additionally expressed with CD gene by these GESTECs, the anticancer activity was significantly increased. In the migration assay, the GESTECs selectively migrated to HeLa cervical cancer cells. As results of real-time PCR, chemoattractant ligands such as MCP-1, SCF, and VEGF were expressed in HeLa cells, and several receptors such as uPAR, VEGFR2, and c-kit were produced by the GESTECs. These GESTECs transduced with CD gene and IFN-ß may provide a potential of a novel gene therapy for anticervical cancer treatments via their selective tumor tropism derived from VEGF and VEGFR2 expressions between HeLa cells and the GESTECs.

Anticancer effect of genistein on BG-1 ovarian cancer growth induced by 17 ß-estradiol or bisphenol A via the suppression of the crosstalk between estrogen receptor α and insulin-like growth factor-1 receptor signaling pathways.

The interaction between estrogen receptor (ER) and insulin-like growth factor-1 receptor (IGF-1R) signaling pathway plays an important role in proliferation of and resistance to endocrine therapy to estrogen dependent cancers. Estrogen (E2) upregulates the expression of components of IGF-1 system and induces the downstream of mitogenic signaling cascades via phosphorylation of insulin receptor substrate-1 (IRS-1). In the present study, we evaluated the xenoestrogenic effect of bisphenol A (BPA) and antiproliferative activity of genistein (GEN) in accordance with the influence on this crosstalk. BPA was determined to affect this crosstalk by upregulating mRNA expressions of ERα and IGF-1R and inducing phosphorylation of IRS-1 and Akt in protein level in BG-1 ovarian cancer cells as E2 did. In the mouse model xenografted with BG-1 cells, BPA significantly increased a tumor burden of mice and expressions of ERα, pIRS-1, and cyclin D1 in tumor mass compared to vehicle, indicating that BPA induces ovarian cancer growth by promoting the crosstalk between ER and IGF-1R signals. On the other hand, GEN effectively reversed estrogenicity of BPA by reversing mRNA and protein expressions of ERα, IGF-1R, pIRS-1, and pAkt induced by BPA in cellular model and also significantly decreased tumor growth and in vivo expressions of ERα, pIRS-1, and pAkt in xenografted mouse model. Also, GEN was confirmed to have an antiproliferative effect by inducing apoptotic signaling cascades. Taken together, these results suggest that GEN effectively reversed the increased proliferation of BG-1 ovarian cancer by suppressing the crosstalk between ERα and IGF-1R signaling pathways upregulated by BPA or E2.

Pancreatic tumor mass in a xenograft mouse model is decreased by treatment with therapeutic stem cells following introduction of therapeutic genes.

Pancreatic cancer is the fourth most common cause of cancer-related mortality. In the present study, we employed 2 types of therapeutic stem cells expressing cytosine deaminase (CD) with or without human interferon-ß (IFN­ß), HB1.F3.CD and HB1.F3.CD.IFN-ß cells, respectively, to selectively treat pancreatic cancer. The CD gene converts the non-toxic prodrug, 5-flurorocytosine (5-FC), into the toxic agent, 5-fluorouracil (5-FU). In addition, human IFN-ß is a potent cytokine that has antitumor effects. To generate a xenograft mouse model, PANC-1 cells (2x10(6)/mouse) cultured in DMEM containing 10% FBS were mixed with Matrigel and were subcutaneously injected into Balb/c nu/nu mice. In the migration assay, the stem cells expressing the CD or IFN-ß gene effectively migrated toward the pancreatic cancer cells, suggesting the presence of chemoattractant factors secreted by the pancreatic tumors. In the co-culture and MTT assay, antitumor activity of the therapeutic stem cells was observed in the presence of 5-FC was shown that the growth of PANC-1 cells was inhibited. Furthermore, these effects were confirmed in the xenograft mouse model bearing tumors originating from PANC-1 cells. Analyses by histological and fluorescence microscopy showed that treatment with the stem cells resulted in the inhibition of pancreatic cancer growth in the presence of 5-FC. Taken together, these results indicate that stem cells expressing the CD and/or IFN-ß gene can be used to effectively treat pancreatic cancer and reduce the side-effects associated with conventional therapies.

Suppression of the growth of human colorectal cancer cells by therapeutic stem cells expressing cytosine deaminase and interferon-ß via their tumor-tropic effect in cellular and xenograft mouse models.

Genetically engineered stem cells (GESTECs) exhibit a potent therapeutic efficacy via their strong tumor tropism toward cancer cells. In this study, we introduced the human parental neural stem cells, HB1.F3, with the human interferon beta (IFN-ß) gene which is a typical cytokine gene that has an antitumor effect and the cytosine deaminase (CD) gene from Escherichia coli (E. coli) that could convert the non-toxic prodrug, 5-fluorocytosine (5-FC), to a toxic metabolite, 5-fluorouracil (5-FU). Two types of stem cells expressing the CD gene (HB1.F3.CD cells) and both the CD and human IFN-ß genes (HB1.F3.CD.IFN-ß) were generated. The present study was performed to examine the migratory and therapeutic effects of these GESTECs against the colorectal cancer cell line, HT-29. When co-cultured with colorectal cancer cells in the presence of 5-FC, HB1.F3.CD and HB1.F3.CD.IFN-ß cells exhibited the cytotoxicity on HT-29 cells via the bystander effect. In particular, HB1.F3.CD.IFN-ß cells showed the synergistic cytotoxic activity of 5-FU and IFN-ß. We also confirmed the migration ability of HB1.F3.CD and HB1.F3.CD.IFN-ß cells toward HT-29 cells by a modified migration assay in vitro, where chemoattractant factors secreted by HT-29 cells attracted the GESTECs. In a xenograft mouse model, the volume of tumor mass was decreased up to 56% in HB1.F3.CD injected mice while the tumor mass was greatly inhibited about 76% in HB1.F3.CD.IFN-ß injected mice. The therapeutic treatment by these GESTECs is a novel strategy where the combination of the migration capacity of stem cells as a vector for therapeutic genes towards colorectal cancer and a synergistic antitumor effect of CD and IFN-ß genes can selectively target this type of cancer.

Benzophenone-1 stimulated the growth of BG-1 ovarian cancer cells by cell cycle regulation via an estrogen receptor alpha-mediated signaling pathway in cellular and xenograft mouse models.

2,4-Dihydroxybenzophenone (benzophenone-1; BP-1) is an UV stabilizer primarily used to prevent polymer degradation and deterioration in quality due to UV irradiation. Recently, BP-1 has been reported to bioaccumulate in human bodies by absorption through the skin and has the potential to induce health problems including endocrine disruption. In the present study, we examined the xenoestrogenic effect of BP-1 on BG-1 human ovarian cancer cells expressing estrogen receptors (ERs) and relevant xenografted animal models in comparison with 17-ß estradiol (E2). In in vitro cell viability assay, BP-1 (10(-8)-10(-5)M) significantly increased BG-1 cell growth the way E2 did. The mechanism underlying the BG-1 cell proliferation was proved to be related with the up-regulation of cyclin D1, a cell cycle progressor, by E2 or BP-1. Both BP-1 and E2 induced cell growth and up-regulation of cyclin D1 were reversed by co-treatment with ICI 182,780, an ER antagonist, suggesting that BP-1 may mediate the cancer cell proliferation via an ER-dependent pathway like E2. On the other hand, the expression of p21, a regulator of cell cycle progression at G1 phase, was not altered by BP-1 though it was down-regulated by E2. In xenograft mouse models transplanted with BG-1 cells, BP-1 or E2 treatment significantly increased the tumor mass formation compared to a vehicle (corn oil) within 8 weeks. In histopathological analysis, the tumor sections of E2 or BP-1 group displayed extensive cell formations with high density and disordered arrangement, which were supported by the increased number of BrdUrd positive nuclei and the over-expression of cyclin D1 protein. Taken together, these results suggest that BP-1 is an endocrine disrupting chemical (EDC) that exerts xenoestrogenic effects by stimulating the proliferation of BG-1 ovarian cancer via ER signaling pathway associated with cell cycle as did E2.

Risk of cardiovascular disease is suppressed by dietary supplementation with protamine and chitooligosaccharide in Sprague-Dawley rats.

Protamine from salmon spermaries is a novel dietary protein. Chitooligosaccharide (COS) is an oligosaccharide derived from chitin or chitosan, a long-chain polymer, by chemical or enzymatic hydrolysis. These two compounds are known to enhance lipid metabolism by interrupting the digestion and absorption of fat in the body. Cardiovascular disease (CVD) refers to any type of specific disease that affects the heart and circulatory system. Dyslipidemia, a condition involving high levels of low-density lipoprotein (LDL) cholesterol and low levels of high-density lipoprotein (HDL) cholesterol, is generally known to be a primary cause of CVD development. The risk of CVD is usually associated with the atherogenic index (AI) and cardiac risk factor (CRF). The CVD risk is also closely associated with serum levels of total cholesterol (T-CHO), LDL cholesterol and HDL cholesterol. In the present study, we evaluated alterations in serum lipid contents following the administration of protamine, COS and mixtures of these two compounds to male Sprague-Dawley (SD) rats, and their ability to reduce CVD risk. Based on the results of a serum lipid assay, protamine, COS and their mixtures were found to significantly reduce AI, CRF and CVD risk by decreasing serum levels of TG, T-CHO and LDL cholesterol and increasing serum HDL cholesterol levels. By contrast, TG and T-CHO concentrations in feces were markedly increased. Accumulation of lipids in the liver tissues of the SD rats fed high-fat diets was also inhibited by the intake of protamine and COS. Our findings suggest that protamine, COS and combinations of the two compounds may be used as a dietary therapy for preventing CVD due to their suppressive effects on hyperlipidemia and hypercholesterolemia.

Genistein, a soy phytoestrogen, prevents the growth of BG-1 ovarian cancer cells induced by 17ß-estradiol or bisphenol A via the inhibition of cell cycle progression.

An endocrine disrupting chemical (EDC) is a global health concern. In this study, we examined the effects of genistein (GEN) on bisphenol A (BPA) or 17ß-estradiol (E2)-induced cell growth and gene alterations of BG-1 ovarian cancer cells expressing estrogen receptors (ERs). In an in vitro cell viability assay, E2 or BPA significantly increased the growth of BG-1 cells. This increased proliferative activity was reversed by treatment with ICI 182,780, a well-known ER antagonist, while cell proliferation was further promoted in the presence of propyl pyrazole triol (PPT), an ERα agonist. These results imply that cell proliferation increased by E2 or BPA was mediated by ERs, particularly ERα. BPA clearly acted as a xenoestrogen in BG-1 ovarian cancer cells by mimicking E2 action. In contrast, GEN effectively suppressed BG-1 cell proliferation promoted by E2 or BPA by inhibiting cell cycle progression. E2 and BPA increased the expression of cyclin D1, a factor responsible for the G1/S cell cycle transition. They also decreased the expression of p21, a potent cyclin-dependent kinase (CDK) inhibitor that arrests the cell cycle in G1 phase, and promoted the proliferation of BG-1 cells. As shown by its repressive effect on cell growth, GEN decreased the expression of cyclin D1 augmented by E2 or BPA. On the other hand, GEN increased the p21 expression downregulated by E2 or BPA. Collectively, our findings suggest that GEN, a dietary phytoestrogen, has an inhibitory effect on the growth of estrogen-dependent cancers promoted by E2 or BPA.

Effects of genetically engineered stem cells expressing cytosine deaminase and interferon-beta or carboxyl esterase on the growth of LNCaP rrostate cancer cells.

The risk of prostate cancer has been increasing in men by degrees. To develop a new prostate cancer therapy, we used a stem cell-derived gene directed prodrug enzyme system using human neural stem cells (hNSCs) that have a tumor-tropic effect. These hNSCs were transduced with the therapeutic genes for bacterial cytosine deaminase (CD), alone or in combination with the one encoding human interferon-beta (IFN-ß) or rabbit carboxyl esterase (CE) to generate HB1.F3.CD, HB1.F3.CD.IFN-ß, and HB1.F3.CE cells, respectively. CD enzyme can convert the prodrug 5-fluorocytosine (5-FC) into the activated form 5-fluorouracil (5-FU). In addition, CE enzyme can convert the prodrug CPT-11 into a toxic agent, SN-38. In our study, the human stem cells were found to migrate toward LNCaP human prostate cancer cells rather than primary cells. This phenomenon may be due to interactions between chemoattractant ligands and receptors, such as VEGF/VEGFR2 and SCF/c-Kit, expressed as cancer and stem cells, respectively. The HB1.F3.CE, HB.F3.CD, or HB1.F3.CD.IFN-ß cells significantly reduced the LNCaP cell viability in the presence of the prodrugs 5-FC or CPT-11. These results indicate that stem cells expressing therapeutic genes can be used to develop a new strategy for selectively treating human prostate cancer.

Therapeutic potential of stem cells expressing suicide genes that selectively target human breast cancer cells: evidence that they exert tumoricidal effects via tumor tropism (review).

Breast cancer is the most prevalent cancer in women worldwide and is classified into ductal and lobular carcinoma. Breast cancer as well as lobular carcinoma is associated with various risk factors such as gender, age, female hormone exposure, ethnicity, family history and genetic risk factor-associated genes. Genes associated with a high risk of developing breast cancer include BRCA1, BRCA2, p53, PTEN, CHEK2 and ATM. Surgery, chemotherapy, radiotherapy and hormone therapy are used to treat breast cancer but these therapies, except for surgery, have many side-effects such as alopecia, anesthesia, diarrhea and arthralgia. Gene-directed enzyme/prodrug therapy (GEPT) or suicide gene therapy, may improve the therapeutic efficacy of conventional cancer radiotherapy and chemotherapy without side-effects. GEPT most often involves the use of a viral vector to deliver a gene not found in mammalian cells and that produces enzymes which can convert a relatively non-toxic prodrug into a toxic agent. Examples of these systems include cytosine deaminase/5-fluorocytosine (CD/5-FC), carboxyl esterase/irinotecan (CE/CPT-11), and thymidine kinase/ganciclovir (TK/GCV). Recently, therapies based on genetically engineered stem cells (GESTECs) using a GEPT system have received a great deal of attention for their clinical and therapeutic potential to treat breast cancer. In this review, we discuss the potential of GESTECs via tumor tropism effects and therapeutic efficacy against several different types of cancer cells. GESTECs represent a useful tool for treating breast cancer without inducing injuries associated with conventional therapeutic modalities.

Modulation of lipid metabolism by mixtures of protamine and chitooligosaccharide through pancreatic lipase inhibitory activity in a rat model.

Overweight and obesity are usually related with high fat and calorie intake, and seriously causative of lifestyle-related diseases such as cardiovascular disorders, arteriosclerosis, and colon cancer. In this study, we propose a novel dietary therapy against overweight and obesity using mixtures of protamine and chitooligosaccharide (COS), which are known to interrupt the lipid metabolism in the body. Protamine is a dietary protein originated from salmon reproductive organ, and COS is an oligosaccharide made from chitin or chitosan by chemical or enzymatic hydrolysis. In the enzyme activity analysis in vitro, protamine and COS strongly suppressed the activity of pancreatic lipase, which is the primary enzyme for the digestion and absorption of lipids in the intestine. In in vivo animal test, the mixtures of protamine and COS significantly reduced the serum levels of triglyceride (TG), total cholesterol (T-CHO), and low density lipoprotein-cholesterol (LDLC) and inhibited the accumulation of lipids in liver tissue of Sprague Dawley (SD) rats fed high fat diets. On the other hand, they increased fecal TG and T-CHO contents. From these alterations in lipid metabolism, we verified that protamine and COS mixtures could effectively interrupt the digestion and absorption of dietary lipids in the body by inhibiting pancreatic lipase activity. In addition, protamine and COS mixtures increased the serum level of high density lipoprotein-cholesterol (HDLC), responsible for removing cholesterol from cells and protecting atherosclerosis, and therefore decreased the potential risks of cardiovascular diseases by lowering values of the atherogenic index (AI) and cardiac risk factor (CRF). Taken together, we suggest protamine and COS mixtures as a prominent dietary therapy for the prevention of overweight, obesity, and further cardiovascular diseases related with hyperlipidemia.