Cheng Wei Lin

Heme oxygenase-1 inhibits breast cancer invasion via suppressing the expression of matrix metalloproteinase-9

In the present study, we investigated the antitumor effects of the invasiveness and migration of heme oxygenase 1 (HO-1) in human breast carcinoma cells. 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced matrix metalloproteinase-9 (MMP-9) enzyme activity and gene expression at both protein and mRNA levels were examined in human breast carcinoma cells (MCF-7 and MDA-MB-231), and the addition of the MMP-9 inhibitor, SB3CT, significantly suppressed TPA-induced invasion and migration according to the in vitro Transwell assay. Elevation of HO-1 gene expression by ferric protoporphyrin IX inhibited TPA-induced invasion of MCF-7 cells, which was blocked by adding the heme oxygenase inhibitor, tin protoporphyrin IX, or transfection of cells with HO-1 short hairpin RNA. MCF-7 cells overexpressing HO-1 (MCF-7/HO-1) were established in the present study, and TPA-induced MMP-9 gene expression, tumor invasion, and colony formation were significantly reduced in MCF-7/HO-1 cells, compared with those in Neo-transfected cells. Activation of protein kinase Cα/extracellular signal-regulated kinases/AP-1 with stimulation of reactive oxygen species production was involved in TPA-induced invasion of MCF-7 cells, which was attenuated by HO-1 protein induced by ferric protoporphyrin IX or transfection of HO-1 expression vectors. Additionally, the addition of carbon monoxide, but not ferric ions, biliverdin, or bilirubin, inhibited TPA-induced invasion through suppressing MMP-9, extracellular signal-regulated kinases, and AP-1 activation stimulated by TPA. The beneficial role of HO-1 in blocking tumor invasion was first identified in this study. [Mol Cancer Ther 2008;7(5):1195–1206]

Gossypol reduction of tumor growth through ROS‐dependent mitochondria pathway in human colorectal carcinoma cells

Among 13 different cell lines, gossypol (GOS) showed the most potent cytotoxic effect against human colorectal carcinoma cells including HT29, COLO205, COLO320HSR and COLO320DM cells according to an MTT assay. The cytotoxic effect of GOS was mediated by its induction of apoptosis as characterized by the occurrence of DNA ladders, apoptotic bodies and chromosome condensation in both COLO205 and HT29 cells. Activation of caspase 3, 6, 8 and 9, but not caspase 1, accompanied by the appearance of cleaved fragments of PARP (85 kDa), and caspase 3 (p17/p15), was identified in GOS‐treated cells. Decreases in Bcl‐xL and phosphorylated Bad proteins were found in GOS‐treated cells. GOS induction of ROS production was detected by in vitro plasmid digestion, and an increase in the intracellular peroxide level was observed in GOS‐treated COLO205 cells by the DCHF‐DA assay. Antioxidants including N‐acetyl‐L‐cysteine (NAC), catalase (CAT), tempol (TEM) and melatonin (MEL), but not allopurinol (ALL), pyrrolidine dithiocarbamate (PDTC) or diphenylene iodonium (DPI), significantly inhibited GOS‐induced Reactive oxygen species (ROS) production through blocking the occurrence of apoptosis. GOS induced mitochondrial dysfunction characterized by a loss of the mitochondria membrane potential via DiOC6 staining, and the release of cytochrome c (Cyt c) and apoptosis‐inducing factor (AIF) from mitochondria to the cytoplasm was observed. Removing mitochondria by ethidium bromide (EtBr) treatment significantly reduced the apoptotic effect of GOS in COLO205 cells. Furthermore, an intraperitoneal injection of GOS or gossypol acetic acid (GAA) significantly reduced the growth of colorectal carcinoma induced by a subcutaneous injection of COLO205 cells in nude mice. Results of the present study provide the first evidences demonstrating the in vitro and in vivo antitumor effects of GOS via an ROS‐dependent mitochondrial apoptosis in colorectal carcinoma.

IGF-I plus E2 induces proliferation via activation of ROS-dependent ERKs and JNKs in human breast carcinoma cells

Induction of 17β‐estradiol (E2) and insulin‐like growth factor‐I (IGF‐I) has been detected in breast carcinoma, however the interaction between E2 and IGF‐I in the proliferation of breast carcinoma cells is still unclear. In the present study, we found that IGF‐I enhances the E2‐induced proliferation in MCF‐7 human breast carcinoma cells in accordance with stimulation of colony formation via a soft agar assay. Activation of insulin receptor substrate‐1 (IRS‐1) protein and extracellular signal‐related kinases (ERKs) and c‐Jun N‐terminal kinases (JNKs), but not p38 mitogen‐activated protein kinase (MAPK), via phosphorylation induction was detected in MCF‐7 cells treated with IGF‐I plus E2 (E2/IGF‐I). E2/IGF‐I‐induced proliferation was blocked by chemical inhibitors of ERKs (PD98059) and JNKs (SP600125). An increase in the expression of c‐Jun protein was detected in E2/IGF‐I‐treated MCF‐7 cells, and this was inhibited by PD98059 and SP600125. Transfection of the dominant negative MEKK and JNK plasmids significantly reduced E2/IGF‐I‐induced proliferation with suppression of c‐Jun protein expression. An increase in peroxide production was detected in E2/IGF‐I‐treated cells, and N‐acetyl‐L‐cysteine (NAC) and Tiron (TIR) addition significantly inhibited E2/IGF‐I‐induced cell proliferation with blocking of the phosphorylation of ERKs and JNKs, and the expression of c‐Jun protein. Additionally, 3‐OH flavone, baicalein, and quercetin showed effective inhibitory activities against E2/IGF‐I‐induced proliferation through suppressing proliferative events such as phosphorylation of IRS‐1, ERKs, and JNKs proteins, and induction of c‐Jun protein and colony formation. These results indicate that IGF‐I interacts with E2 to promote the proliferation of breast carcinoma cells via ROS‐dependent MAPK activation and c‐Jun protein expression. The structure‐related inhibition of E2/IGF‐I‐induced proliferative events by flavonoids is elucidated. J. Cell. Physiol. 212:666–674, 2007.

Flavonoids Luteolin and Quercetin Inhibit RPS19 and contributes to metastasis of cancer cells through c-Myc reduction

Flavonoids luteolin and quercetin can inhibit growth and metastasis of cancer cells. In our previous report, luteolin and quercetin was shown to block Akt/mTOR/c-Myc signaling. Here, we found luteolin and quercetin reduced protein level and transactivation activity of RPS19 in A431-III cells, which is isolated from parental A431 (A431-P) cell line. Further investigation the inhibitory mechanism of luteolin and quercetin on RPS19, we found c-Myc binding sites on RPS19 promoter. The Akt inhibitor LY294002, mTOR inhibitor rapamycin and c-Myc inhibitor 10058-F4 significantly suppressed RPS19 expression and transactivation activities. Overexpression and knockdown of c-Myc in cancer cells show RPS19 expression was regulated by c-Myc. Furthermore, Knockdown and overexpression of RPS19 was used to analyze of the function of RPS19 in cancer cells. The epithelial-mesenchymal transition (EMT) markers and metastasis abilities of cancer cells were also regulated by RPS19. These data suggest that luteolin and quercetin might inhibit metastasis of cancer cells by blocking Akt/mTOR/c-Myc signaling pathway to suppress RPS19-activated EMT signaling.

Activation of fibroblasts by nicotine promotes the epithelial-mesenchymal transition and motility of breast cancer cells

The tumor microenvironment plays an important role in tumor initiation and progression. It is well documented that nicotine participates in cigarette smoking‐related malignancies. Previous studies focused on the effects of nicotine on tumor cells; however, the role of the microenvironment in nicotine‐mediated tumorigenesis is poorly understood. Herein, we investigated the effect and molecular mechanism of nicotine on fibroblasts and its contribution to breast cancer. We found that nicotine induced the epithelial‐mesenchymal transition (EMT) of breast cancer cells and promoted activation of fibroblasts. Interestingly, conditioned medium from nicotine‐activated fibroblasts (Nic–CM) had a greater impact on promoting the EMT and migratory capability toward cancer cells than did treatment with nicotine alone. Production of connective tissue growth factor (CTGF) and transforming growth factor (TGF)‐β by nicotine‐treated fibroblasts was demonstrated to be crucial for promoting the EMT and cancer cell migration, and blocking of CTGF and TGF‐β in Nic‐CM‐suppressed tumor motility. Moreover, nicotine induced expressions of CTGF, and TGF‐β in fibroblasts as identified through α7 nicotinic acetylcholine receptor (nAChR)‐dependent activation of the AKT/TAZ signaling mechanism. Together, our data showed for the first time that activation of fibroblasts is largely responsible for accelerating smoking‐mediated breast cancer progression.

Panobinostat sensitizes KRAS-mutant non-small-cell lung cancer to gefitinib by targeting TAZ

Mutation of KRAS in non‐small‐cell lung cancer (NSCLC) shows a poor response to epidermal growth factor receptor (EGFR) inhibitors and chemotherapy. Currently, there are no direct anti‐KRAS therapies available. Thus, new strategies have emerged for targeting KRAS downstream signaling. Panobinostat is a clinically available histone deacetylase inhibitor for treating myelomas and also shows potentiality in NSCLC. However, the therapeutic efficacy of panobinostat against gefitinib‐resistant NSCLC is unclear. In this study, we demonstrated that panobinostat overcame resistance to gefitinib in KRAS‐mutant/EGFR‐wild‐type NSCLC. Combined panobinostat and gefitinib synergistically reduced tumor growth in vitro and in vivo. Mechanistically, we identified that panobinostat—but not gefitinib—inhibited TAZ transcription, and the combination of panobinostat and gefitinib synergistically downregulated TAZ and TAZ downstream targets, including EGFR and EGFR ligand. Inhibition of TAZ by panobinostat or short hairpin RNA sensitized KRAS‐mutant/EGFR‐wild‐type NSCLC to gefitinib through abrogating AKT/mammalian target of rapamycin (mTOR) signaling. Clinically, TAZ was positively correlated with EGFR signaling, and coexpression of TAZ/EGFR conferred a poorer prognosis in lung cancer patients. Our findings identify that targeting TAZ‐mediated compensatory mechanism is a novel therapeutic approach to overcome gefitinib resistance in KRAS‐mutant/EGFR‐wild‐type NSCLC.