Jyh Ming Chow

Inhibition of MDA-MB-231 breast cancer cell proliferation and tumor growth by apigenin through induction of G2/M arrest and histone H3 acetylation-mediated p21WAF1/CIP1 expression

Apigenin (4′,5,7‐trihydroxyflavone), a flavonoid commonly found in fruits and vegetables, has anticancer properties in various malignant cancer cells. However, the molecular basis of the anticancer effect remains to be elucidated. In this study, we investigated the cellular mechanisms underlying the induction of cell cycle arrest by apigenin. Our results showed that apigenin at the nonapoptotic induction concentration inhibited cell proliferation and induced cell cycle arrest at the G2/M phase in the MDA‐MB‐231 breast cancer cell line. Immunoblot analysis indicated that apigenin suppressed the expression of cyclin A, cyclin B, and cyclin‐dependent kinase‐1 (CDK1), which control the G2‐to‐M phase transition in the cell cycle. In addition, apigenin upregulated p21WAF1/CIP1 and increased the interaction of p21WAF1/CIP1 with proliferating cell nuclear antigen (PCNA), which inhibits cell cycle progression. Furthermore, apigenin significantly inhibited histone deacetylase (HDAC) activity and induced histone H3 acetylation. The subsequent chromatin immunoprecipitation (ChIP) assay indicated that apigenin increased acetylation of histone H3 in the p21WAF1/CIP1 promoter region, resulting in the increase of p21WAF1/CIP1 transcription. In a tumor xenograft model, apigenin effectively delayed tumor growth. In these apigenin‐treated tumors, we also observed reductions in the levels of cyclin A and cyclin B and increases in the levels of p21WAF1/CIP1 and acetylated histone H3. These findings demonstrate for the first time that apigenin can be used in breast cancer prevention and treatment through epigenetic regulation.

Nonautophagic cytoplasmic vacuolation death induction in human PC-3M prostate cancer by curcumin through reactive oxygen species -mediated endoplasmic reticulum stress

The antiapoptotic and antiautophagic abilities of cancer cells constitute a major challenge for anticancer drug treatment. Strategies for triggering nonapoptotic or nonautophagic cell death may improve therapeutic efficacy against cancer. Curcumin has been reported to exhibit cancer chemopreventive properties. Herein, we report that curcumin induced apoptosis in LNCaP, DU145, and PC-3 cells but triggered extensive cytoplasmic vacuolation in PC-3M cells. Electron microscopic images showed that the vacuoles lacked intracellular organelles and were derived from the endoplasmic reticulum (ER). Moreover, curcumin-induced vacuolation was not reversed by an apoptosis- or autophagy-related inhibitor, suggesting that vacuolation-mediated cell death differs from classical apoptotic and autophagic cell death. Mechanistic investigations revealed that curcumin treatment upregulated the ER stress markers CHOP and Bip/GRP78 and the autophagic marker LC3-II. In addition, curcumin induced ER stress by triggering ROS generation, which was supported by the finding that treating cells with the antioxidant NAC alleviated curcumin-mediated ER stress and vacuolation-mediated death. An in vivo PC-3M orthotopic prostate cancer model revealed that curcumin reduced tumor growth by inducing ROS production followed by vacuolation-mediated cell death. Overall, our results indicated that curcumin acts as an inducer of ROS production, which leads to nonapoptotic and nonautophagic cell death via increased ER stress.

Lipocalin 2 prevents oral cancer metastasis through carbonic anhydrase IX inhibition and is associated with favourable prognosis

Lipocalin 2 (LCN2), a secreted glycoprotein, is up- or downregulated in different human cancers. At present, the functional role of LCN2 in the progression of oral squamous cell carcinoma (OSCC), which accounts for most head and neck cancers, remains poorly understood, particularly with respect to its involvement in invasion and metastasis. In this study, we observed that LCN2 expression decreased in patients with OSCC and lymph node metastasis compared with that in patients without metastasis. A higher LCN2 expression correlated with the survival of patients with OSCC. Furthermore, LCN2 overexpression in OSCC cells reduced in vitro migration and invasion and in vivo metastasis, whereas its silencing induced an increase in cell motility. Mechanistically, LCN2 inhibited the cell motility of OSCC cells through hypoxia-inducible factor (HIF)-1α-dependent transcriptional inhibition of the carbonic anhydrase IX (CAIX). CAIX overexpression relieved the migration inhibition imposed by LCN2 overexpression in OSCC cells. Moreover, a microRNA (miR) analysis revealed that LCN2 can suppress CAIX expression and cell migration through miR-4505 induction. Examination of tumour tissues from patients with OSCC and OSCC-transplanted mice revealed an inverse correlation between LCN2 and CAIX expression. Furthermore, patients with LCN2(strong)/CAIX(weak) revealed the lowest frequency of lymph node metastasis and the longest survival. Our findings suggest that LCN2 suppresses tumour metastasis by targeting the transcriptional and post-transcriptional regulation of CAIX in OSCC cells. LCN2 overexpression may be a novel OSCC treatment strategy and a useful biomarker for predicting OSCC progression.

Suppression of reactive oxygen species-mediated ERK and JNK activation sensitizes dihydromyricetin-induced mitochondrial apoptosis in human non-small cell lung cancer.

Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer with a high mortality rate and still remains a therapeutic challenge. A strategy for targeting NSCLC is to identify agents that are effective against NSCLC cells while sparing normal cells. Dihydromyricetin (DHM) is the major flavonoid component derived from Ampelopsis grossedentata, which has a long history of use in medicine. Herein, the molecular mechanisms by which DHM exerts its anticancer effects against NSCLC cells were investigated. Results from MTS, colony formation, Western blot, flow cytometric, and JC‐1 mitochondrial membrane potential assays revealed that DHM showed a selective cytotoxic effect against NSCLC cells (A549 and H1975), but not against normal lung (WI‐38) fibroblasts, by inducing apoptosis. DHM‐induced cell apoptosis occurred through Bcl‐w suppression‐mediated mitochondrial membrane depolarization, caspase‐9/‐7/‐3 activation, and poly(ADP‐ribose) polymerase (PARP) cleavage in A549 and H1975 cells. Moreover, treatment of A549 and H1975 cells with DHM induced increase of intracellular peroxide and sustained activation of extracellular signal‐regulated kinase (ERK)1/2 and c‐Jun N‐terminal kinase (JNK)1/2, and the reactive oxygen species scavenger, N‐acetylcysteine (NAC), reversed DHM‐induced ERK and JNK activation. Furthermore, treatment of cells with specific inhibitors of ERK and JNK or NAC significantly promoted the DHM‐induced activation of caspase‐9/‐7/‐3 and PARP cleavage and also sensitized the antitumorigenic effect of DHM on NSCLC cells. These findings define and support a novel function of DHM of inducing mitochondrion‐derived apoptosis in human NSCLC cells, and a combination of DHM with ERK and JNK inhibitors should be a good strategy for preventing NSCLC proliferation.

Keap1-Nrf2 Interaction Suppresses Cell Motility in Lung Adenocarcinomas by Targeting the S100P Protein.

Purpose: Kelch-like ECH-associated protein 1 (Keap1) is an E3 ligase participated in the cellular defense response against oxidative stress through nuclear factor erythroid-2–related factor 2 (Nrf2). However, the role of Keap1 in regulating cancer motility is still controversial. We investigated the contribution of the Keap1–Nrf2 axis in the progression of non–small cell lung cancer (NSCLC). Experimental Design: The expression of Keap1 and Nrf2 was examined via immunohistochemistry, real-time PCR, and Western blot analysis in a cohort of NSCLC tissues and cells. A series of in vivo and in vitro assays was performed to elucidate the contribution of the Keap1–Nrf2 axis in lung cancer mobility and progression. Results: Keap1 expression was decreased in specimens from NSCLC patients with lymph node metastasis compared with patients without metastasis. Higher Keap1 expression levels were correlated with the survival of NSCLC patients. Moreover, manipulation of Keap1 expression affected cell migration/invasion abilities. Depletion of Nrf2 relieved the migration promotion imposed by Keap1 suppression. Mechanistic investigations found that S100P was downregulated in both Keap1-overexpressing and Nrf2-knockdown NSCLC cells. Overexpression of Keap1 and knockdown of Nrf2 both suppressed S100P expression in NSCLC cells. Knockdown of S100P inhibited cell migration in highly invasive NSCLC cells and also relieved the migration promotion imposed by Keap1 suppression in weakly invasive NSCLC cells. Conclusions: Our findings suggest that Keap1 functions as a suppressor of tumor metastasis by targeting the Nrf2/S100P pathway in NSCLC cells. In addition, overexpression of Keap1 may be a novel NSCLC treatment strategy and/or useful biomarker for predicting NSCLC progression. Clin Cancer Res; 21(20); 4719–32. ©2015 AACR.

Tricetin suppresses the migration/invasion of human glioblastoma multiforme cells by inhibiting matrix metalloproteinase-2 through modulation of the expression and transcriptional activity of specificity protein 1.

Objective: Glioblastoma multiforme (GBM) is a severely invasive tumor that can be fatal because it is difficult to treat. Tricetin, a natural flavonoid, was demonstrated to inhibit the growth of various cancers, but the effect of tricetin on cancer motility is largely unknown. Research design and methods: In the present study, we examined the anti-invasive properties of tricetin in huwman GBM cells. Results: Our results showed that tricetin inhibited the migration/invasion of two GBM cell lines. We found that tricetin inhibited MMP-2 expression in the GBM cells. Real-time polymerase chain reaction and promoter activity assays indicated that tricetin inhibited MMP-2 expression at the transcriptional level. Such inhibitory effects were associated with the suppression of specificity protein-1 (SP-1) DNA-binding activity. An examination of clinical samples revealed a positive correlation between SP-1 and MMP-2 in glioma specimens, and higher expression levels were correlated with a worse probability of survival. Moreover, blocking the extracellular signal-regulated kinase (ERK) pathway also inhibited MMP-2-mediated cell motility, and further enhanced the anti-invasive ability of tricetin in GBM cells. Conclusions: SP-1 is an important target of tricetin for suppressing MMP-2-mediated cell motility in GBM cells, and a combination of tricetin and an ERK inhibitor may be a good strategy for preventing GBM invasion.

Quercetin simultaneously induces G0/G1‐phase arrest and caspase‐mediated crosstalk between apoptosis and autophagy in human leukemia HL‐60 cells

Quercetin is a plant‐derived bioflavonoid with high anticancer activity in various tumors. Herein, the molecular mechanisms by which quercetin exerts its anticancer effects against HL‐60 acute myeloid leukemia (AML) cells were investigated. Results showed that quercetin suppressed cell proliferation in the HL‐60 cell line in vitro and in vivo. Quercetin‐induced G0/G1‐phase arrest occurred when expressions of cyclin‐dependent kinase (CDK)2/4 were inhibited and the CDK inhibitors, p16 and p21, were induced. Moreover, quercetin treatment not only activated proapoptotic signaling like poly (ADP ribose) polymerase (PARP)−1 cleavage and caspase activation but also triggered autophagy events as shown by the increased expression of light chain 3 (LC3)‐II, decreased expression of p62, and formation of acidic vesicular organelles. Interestingly, it was found that use of the autophagy inhibitor, 3‐methyladenine, significantly enhanced quercetin‐mediated apoptotic cell death as analyzed by MTS and DNA fragmentation assays. Moreover, pretreatment of HL‐60 cells with the pan‐caspase inhibitor, Z‐VAD‐fmk, dramatically reversed quercetin‐mediated apoptotic and autophagic cell death. Although apoptosis and autophagy are two independent cell death pathways, our findings indicated that quercetin can activate caspases to trigger these two pathways, and both pathways played contrary roles in quercetin‐mediated HL‐60 cell death. In conclusion, besides promoting apoptosis, quercetin also induced cytoprotective autophagy in HL‐60 cells, and inhibition of autophagy may be a novel strategy to enhance the anticancer activity of quercetin in AML.

Tricetin Induces Apoptosis of Human Leukemic HL-60 Cells through a Reactive Oxygen Species-Mediated c-Jun N-Terminal Kinase Activation Pathway

Tricetin is a dietary flavonoid with cytostatic properties and antimetastatic activities in various solid tumors. The anticancer effect of tricetin in nonsolid tumors remains unclear. Herein, the molecular mechanisms by which tricetin exerts its anticancer effects on acute myeloid leukemia (AML) cells were investigated. Results showed that tricetin inhibited cell viability in various types of AML cell lines. Tricetin induced morphological features of apoptosis such as chromatin condensation and phosphatidylserine (PS) externalization, and significantly activated proapoptotic signaling including caspase-8, -9, and -3 activation and poly(ADP-ribose) polymerase (PARP) cleavage in HL-60 AML cells. Of note, tricetin-induced cell growth inhibition was dramatically reversed by a pan caspase and caspase-8- and -9-specific inhibitors, suggesting that this compound mainly acts through a caspase-dependent pathway. Moreover, treatment of HL-60 cells with tricetin induced sustained activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), and inhibition of ERK and JNK by their specific inhibitors respectively promoted and abolished tricetin-induced cell apoptosis. Dichlorofluorescein (DCF) staining showed that intracellular reactive oxygen species (ROS) levels were higher in tricetin-treated HL-60 cells compared to the control group. Moreover, an ROS scavenger, N-acetylcysteine (NAC), reversed tricetin-induced JNK activation and subsequent cell apoptosis. In conclusion, our results indicated that tricetin induced cell death of leukemic HL-60 cells through induction of intracellular oxidative stress following activation of a JNK-mediated apoptosis pathway. A combination of tricetin and an ERK inhibitor may be a better strategy to enhance the anticancer activities of tricetin in AML.

Trichodermin induces c-Jun N-terminal kinase-dependent apoptosis caused by mitotic arrest and DNA damage in human p53-mutated pancreatic cancer cells and xenografts

Pancreatic cancer is an aggressive malignancy, which generally responds poorly to chemotherapy. In this study, trichodermin, an endophytic fungal metabolite from Nalanthamala psidii, was identified as a potent and selective antitumor agent in human pancreatic cancer. Trichodermin exhibited antiproliferative effects against pancreatic cancer cells, especially p53-mutated cells (MIA PaCa-2 and BxPC-3) rather than normal pancreatic epithelial cells. We found that trichodermin induced caspase-dependent and mitochondrial intrinsic apoptosis. Trichodermin also increased apoptosis through mitotic arrest by activating Cdc2/cyclin B1 complex activity. Moreover, trichodermin promoted the activation of c-Jun N-terminal kinase (JNK), and inhibition of JNK by its inhibitor, shRNA, or siRNA significantly reversed trichodermin-mediated caspase-dependent apoptosis. Trichodermin triggered DNA damage stress to activate p53 function for executing apoptosis in p53-mutated cells. Importantly, we demonstrated that trichodermin with efficacy similar to gemcitabine, profoundly suppressed tumor growth through inducing intratumoral DNA damage and JNK activation in orthotopic pancreatic cancer model. Based on these findings, trichodermin is a potential therapeutic agent worthy of further development into a clinical trial candidate for treating cancer, especially the mutant p53 pancreatic cancer.

Activation of p53/miR-34a Tumor Suppressor Axis by Chinese Herbal Formula JP-1 in A549 Lung Adenocarcinoma Cells

Lung cancer is the leading cause of cancer death worldwide; the most common pathologic type is lung adenocarcinoma (LADC). In spite of the recent progress in targeted therapy, most LADC patients eventually expired due to the inevitable recurrence and drug resistance. New complementary agent with evidence-based molecular mechanism is urgently needed. MiR-34a is an important p53 downstream tumor suppressor, which regulates apoptosis, cell-cycle, EMT (epithelial mesenchymal transition), and so forth. Its expression is deficient in many types of cancers including LADC. Here, we show that a Chinese herbal formula JP-1 activates p53/miR-34a axis in A549 human LADC cells (p53 wild-type). Treatment with JP-1 induces p53 and its downstream p21 and BAX proteins as well as the miR-34a, resulting in growth inhibition, colony formation reduction, migration repression, and apoptosis induction. Accordingly, the decreases of miR-34a downstream targets such as CDK6, SIRT1, c-Myc, survivin, Snail, and AXL were observed. Moreover, JP-1 activates AMPKα and reduces mTOR activity, implying its inhibitory effect on the energy-sensitive protein synthesis and cell proliferation signaling. Our results show that JP-1 activates p53/miR-34a tumor suppressor axis and decreases proteins related to proliferation, apoptosis resistance, and metastasis, suggesting its potential as a complementary medicine for LADC treatment.