Li-Ching Chen

Nicotine-induced human breast cancer cell proliferation attenuated by garcinol through down-regulation of the nicotinic receptor and cyclin D3 proteins

Previous studies have demonstrated that the persistent exposure of human bronchial epithelial cells to nicotine (Nic) through nicotinic acetylcholine receptors increases cyclin D1 promoter activity and protein expression. The main purpose of this study is to elucidate the carcinogenic role of cyclin D3, which is involved in breast tumorigenesis when induced by Nic. Real-time PCR analysis revealed that cyclin D3 is highly expressed at the mRNA level in surgically dissected breast tumor tissue, compared to the surrounding normal tissue (tumor/normal fold ratioxa0=xa017.93, nxa0=xa074). To test whether Nic/nicotinic acetylcholine receptor (nAChR) binding could affect cyclin D3 expression in human breast cancer cells, the transformed cell line MCF-10A-Nic (DOX) was generated from normal breast epithelial cells (MCF-10A) with inducible α9-nAChR gene expression, using the adenovirus tetracycline-regulated Tet-off system. Tet-regulated overexpression of α9-nAChR in MCF-10A-Nic (DOX) xenografted BALB/c-nu/nu mice resulted in a significant induction of cyclin D3. In contrast, cyclin D3 expression was down-regulated in α9-nAChR knock-down (siRNA) MDA-MB-231-xenografted tumors in NOD.CB17-PRKDC(SCID)/J(NOD-SCID) mice. Furthermore, we found that Nic-induced human breast cancer (MDA-MB-231) cell proliferation was inhibited by 1xa0μM of garcinol (Gar), isolated from the edible fruit Garcinia indica, through down-regulation of α9-nAChR and cyclin D3 expression. These results suggest that α9-nAChR-mediated cyclin D3 overexpression is important for nicotine-induced transformation of normal human breast epithelial cells. The homeostatic regulation of cyclin D3 has the potential to be a molecular target for antitumor chemotherapeutic or chemopreventive purposes in clinical breast cancer patients.

Inhibition of anchorage-independent proliferation and G0/G1 cell-cycle regulation in human colorectal carcinoma cells by 4,7-dimethoxy-5-methyl-l,3- benzodioxole isolated from the fruiting body of antrodia camphorate

In this study, 4,7-dimethoxy-5-methyl-l,3-benzodioxole (SY-1) was isolated from three different sources of dried fruiting bodies of Antrodia camphorate (AC). AC is a medicinal mushroom that grows on the inner heartwood wall of Cinnamomum kanehirai Hay (Lauraceae), an endemic species that is used in Chinese medicine for its anti-tumor and immunomodulatory properties. In this study, we demonstrated that SY-1 profoundly decreased the proliferation of human colon cancer cells (COLO 205) through G0/G1 cell-cycle arrest (50–150u2009μM) and induction of apoptosis (>150u2009μM). Cell-cycle arrest induced by SY-1 was associated with a significant increase in levels of p53, p21/Cip1 and p27/Kip1, and a decrease in cyclins D1, D3 and A. In contrast, SY-1 treatment did not induce significant changes in G0/G1 phase cell-cycle regulatory proteins in normal human colonic epithelial cells (FHC). The cells were cultured in soft agar to evaluate anchorage-independent colony formation, and we found that the number of transformed colonies was significantly reduced in the SY-1-treated COLO 205 cells. These findings demonstrate for the first time that SY-1 inhibits human colon cancer cell proliferation through inhibition of cell growth and anchorage-independent colony formation in soft agar. However, the detailed mechanisms of these processes remain unclear and will require further investigation.

An apple a day to prevent cancer formation: Reducing cancer risk with flavonoids

The purpose of this review is to update and discuss key findings from inxa0vitro and inxa0vivo studies on apple and its biocompounds, with a special focus on its anticancer role. Several studies have proposed that apple and its extracts exhibit a variety of biological functions that may contribute to health benefits including beneficial effects against chronic heart and vascular disorders, respiratory and pulmonary dysfunction, diabetes, obesity, and cancer. In this review, we summarize the molecular mechanism(s) of various components in apple, as established in previous studies that indicated their growth-inhibitory effects in various cancer cell types. Moreover, an attempt is made to delineate the direction of future studies that could lead to the development of apple components as a potent chemopreventive/chemotherapeutic agent against cancer.

Protein phosphatase Mg2+/Mn2+ dependent 1F promotes smoking-induced breast cancer by inactivating phosphorylated-p53-induced signals

We previously demonstrated that the activation of α9-nicotinic acetylcholine receptor (α9-nAchR) signaling by smoking promotes breast cancer formation. To investigate the downstream signaling molecules involved in α9-nAChR-induced breast tumorigenesis, we used real-time polymerase chain reactions and Western blotting to assess expression of protein phosphatase Mg2+/Mn2+ dependent 1F (PPM1F), a Ser/Thr protein phosphatase, in human breast cancer samples (n=167). Additionally, stable PPM1F-knockdown and -overexpressing cell lines were established to evaluate the function of PPM1F. The phosphatase activity of PPM1F in nicotine-treated cells was assessed through Western blotting, confocal microscopy, and fluorescence resonance energy transfer. Higher levels of PPM1F were detected in the breast cancer tissues of heavy smokers (n=7, 12.8-fold) greater than of non-smokers (n= 28, 6.3-fold) (**p=0.01). In vitro, nicotine induced PPM1F expression, whereas α9-nAChR knockdown reduced the protein expression of PPM1F. A series of biochemical experiments using nicotine-treated cells suggested that the dephosphorylation of p53 (Ser-20) and BAX (Ser-184) by PPM1F is a critical posttranslational modification, as observed in breast cancer patients who were heavy smokers. These observations indicate that PPM1F may be a mediator downstream of α9-nAChR that activates smoking-induced carcinogenic signals. Thus, PPM1F expression could be used for prognostic diagnosis or inhibited for cancer prevention and therapy.

Long-term ethanol exposure causes human liver cancer cells to become resistant to mitomycin C treatment through the inactivation of bad-mediated apoptosis

The aim of this study was to test whether long‐term ethanol consumption confers therapeutic resistance to human liver cancer patients infected with hepatitis B virus (HBV). Chronic ethanol‐treated cells were established by consecutively culturing a human hepatocellular carcinoma cell line, Hep 3B, which contains integrated HBV sequences, for 20–40 passages with or without 10u2009mM ethanol (designated as E20–E40 and C20–C40, respectively). Flow cytometry analysis demonstrated that a growth promoting effect of long‐term ethanol treatment was induced in the E40 cells through preferential acceleration of S‐phase in these cells. Lower protein expression levels of p16, p21/Cip1, and p27/Kip1 were detected in the ethanol‐treated E40 cells. We further demonstrated that long‐term ethanol‐treated E40 cells develop drug resistance in response to mitomycin C (MMC) treatment (>8u2009µM). Immunoblot analysis revealed that caspase‐8‐mediated mitochondrial apoptotic signals (such as Bad) were inactivated in the MMC‐resistant E40 cells. Immunoprecipitation experiments demonstrated that the sequestration of phosphorylated Bad (Ser‐112) through its binding with 14‐3‐3 was detected more profoundly in the MMC‐resistant E40 cells. Next, we examined the therapeutic efficacy of MMC (10u2009mg MMC/kg body weight, three times per week) in severe combined immunodeficient (SCID) mice bearing E40‐ and C40‐xenografted tumors. Significant reductions (>3‐fold) in tumor growth were detected in MMC‐treated C40‐xenografted mice. In vivo and in vitro studies demonstrated that AKT‐ and extracellular signal‐regulated kinase (ERK)‐mediated survival factors inhibited the Bad‐induced mitochondrial apoptotic signals that were involved in E40 tumor cells and that conferred resistance to MMC.

DNA primase polypeptide 1 (PRIM1) involves in estrogen-induced breast cancer formation through activation of the G2/M cell cycle checkpoint: DNA primase polypeptide 1 (PRIM1) involved in estrogen-induced breast cancer formation

The DNA primase polypeptide 1 (PRIM1) is responsible for synthesizing small RNA primers for Okazaki fragments generated during discontinuous DNA replication. PRIM1 mRNA expression levels in breast tumor samples were detected by real‐time PCR analysis. Xenografted tumor model was established to study the carcinogenic role of PRIM1 and its potential therapeutic applications. The average PRIM1 mRNA (copy number × 103/μg) expression was 4.7‐fold higher in tumors than in normal tissue (*p = 0.005, n = 254). PRIM1 was detected preferentially at a higher level (>40‐fold) in poorly differentiated tumor tissues (n = 46) compared with more highly differentiated tumors tissues (n = 10) (*p = 0.005). Poor overall survival rate was correlated to the estrogen receptor positive (ER+, n = 20) patients with higher PRIM1 expression when compare to the ER− (n = 10) patients (Chi Square test, p = 0.03). Stable expression of PRIM1‐siRNA in the ER+ BT‐474 cells‐xenograft tumors significantly reduced tumor volume in SCID mice (*p = 0.005). The anti‐tumoral effects of inotilone isolated from Phellinus linteus was tested and had significant effects on the inhibition of PRIM1 protein expression in ER+ breast cancer cells. In vivo study was performed by administering inotilone (10 mg/kg, twice a week for 6 weeks), which resulted in significantly reduced BT‐474‐xenografted tumor growth volume compared with control (n =5 per group, *p < 0.05). This study provides evidences for the prognostic effects of PRIM1 with poor overall survival rate in the ER+ patients and will be valuable to test for therapeutic purpose.

The impact of the effectiveness of GATA3 as a prognostic factor in breast cancer

The transcription factor GATA3 plays a significant role in mammary gland development and differentiation. We analyzed expression of GATA3 in breast cancer (BC) cell lines and clinical specimens from BC patients in Taiwan. Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR were carried out to determine the mRNA level of GATA3 from 241 pairs of matched tumor and adjacent normal tissues from anonymous female donors. GATA3 immunohistochemistry (IHC) staining and H-score were performed (n = 25). Inducing and silencing of GATA3 were done by exposure MCF-7 cell line to nicotine or curcumin, respectively. GATA3 expression was detected in most of the estrogen receptor-positive (ER+) tumor specimens (176/241, 73%) compared with paired normal tissues (65/241, 27%) (P < .001). The GATA3 level was highest in Luminal A, and independent t-tests revealed higher GATA3 was associated with ER+ (P = .018) and BC stages (stage II, and stage IV). Nuclear protein expression of GATA3 was detected in tumor tissues (P < .001) with higher H-score in Luminal A patients (P = .012). Kaplan-Meier survival analyses showed that ER+/progesterone receptor (PgR)+ and lower grade BC patients with relatively high GATA3 had better clinical overall survival (OS). GATA3 regulates ERα and BCL-2 as BC luminal subtype markers. Cox univariate and multivariate analyses demonstrated that the expression of GATA3 was an effective predictor of the risk of death. We demonstrated a correlation between GATA3 expression and only ER+ and suggest that a higher GATA3 expression is a good prognostic factor for OS for ER+ BC patients.

Long-term exposure to extremely low-dose of nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induce non-malignant breast epithelial cell transformation through activation of the a9-nicotinic acetylcholine receptor-mediated signaling pathway.

Breast cancer (BC) is the most common cancer affecting women worldwide and has been associated with active tobacco smoking. Low levels of nicotine (Nic) and 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK), have been detected in cases of second‐hand smoke (SHS). However, the correlation between SHS and BC risk remains controversial. In this study, we investigated whether the physiological SHS achievable dose of Nic and tobacco specific nitrosamine, NNK act together to induce breast carcinogenesis using an in vitro breast cell carcinogenesis model. Immortalized non‐tumorigenic breast epithelial cell line, HBL‐100 used for a time‐course assay, was exposed to very low levels of either Nic or NNK, or both. The time‐course assay consisted of 23 cycles of nitrosamines treatment. In each cycle, HBL‐100 cells were exposed to 1pM of Nic and/or 100 femtM of NNK for 48u2009hours. Cells were passaged every 3 days and harvested after 10, 15, and 23 cycles. Our results demonstrated that the tumorigenicity of HBL‐100, defined by soft agar colony forming, proliferation, migration and invasion abilities, was enhanced by co‐exposure to physiologically SHS achievable doses of Nic and NNK. In addition, α9‐nAChR signaling activation, which plays an important role in cellular proliferation and cell survival, was also observed. Importantly, an increase in stemness properties including the prevalence of CD44+/CD24− cells, increase Nanog expression and mammosphere‐forming ability were also observed. Our results indicate that chronic and long term exposure to environmental tobacco smoke, may induce breast cell carcinogenesis even at extremely low doses.