Helicobacter pylori infection induces STAT3 phosphorylation on Ser727 and autophagy in human gastric epithelial cells and mouse stomach

© 2020, The Author(s).Helicobacter pylori (H. pylori) infection is considered as one of the principal risk factors of gastric cancer. Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) plays an important role in inflammation-associated gastric carcinogenesis. In the canonical STAT3 pathway, phosphorylation of STAT3 on Tyr705 is a major event of STAT3 activation. However, recent studies have demonstrated that STAT3 phosphorylated on Ser727 has an independent function in mitochondria. In the present study, we found that human gastric epithelial AGS cells infected with H. pylori resulted in localization of STAT3 phosphorylated on Ser727 (P-STAT3Ser727), predominantly in the mitochondria. Notably, H. pylori-infected AGS cells exhibited the loss of mitochondrial integrity and increased expression of the microtubule-associated protein light chain 3 (LC3), the autophagosomal membrane-associated protein. Treatment of AGS cells with a mitophagy inducer, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), resulted in accumulation of P-STAT3Ser727 in mitochondria. In addition, the elevated expression and mitochondrial localization of LC3 induced by H. pylori infection were attenuated in AGS cells harboring STAT3 mutation defective in Ser727 phosphorylation (S727A). We also observed that both P-STAT3Ser727 expression and LC3 accumulation were increased in the mitochondria of H. pylori-inoculated mouse stomach.

The positive feedback loop between Nrf2 and phosphogluconate dehydrogenase stimulates proliferation and clonogenicity of human hepatoma cells

© 2020, © 2020 Informa UK Limited, trading as Taylor & Francis Group.Recent studies report that nuclear factor-erythroid-2-related factor 2 (Nrf2) facilitates tumor progression through metabolic reprogramming in cancer cells. However, the molecular mechanism underlying the oncogenic functions of Nrf2 is not yet well understood. Some of the pentose phosphate pathway (PPP) enzymes are considered to play a role in the cancer progression. The present study was intended to explore the potential role of phosphogluconate dehydrogenase (PGD), one of the PPP enzymes, in the proliferation and migration of human hepatoma HepG2 cells. Genetic ablation of Nrf2 attenuated the expression of PGD at both transcriptional and translational levels. Notably, Nrf2 regulates the transcription of PGD through direct binding to the antioxidant response element in its promoter region. Nrf2 overexpression in HepG2 cells led to increased proliferation, survival, and migration, and these events were suppressed by silencing PGD. Interestingly, knockdown of the gene encoding this enzyme not only attenuated the proliferation and clonogenicity of HepG2 cells but also downregulated the expression of Nrf2. Thus, there seems to exist a positive feedback loop between Nrf2 and PGD which is exploited by hepatoma cells for their proliferation and survival. Treatment of HepG2 cells with ribulose-5-phosphate, a catalytic product of PGD, gave rise to a concentration-dependent upregulation of Nrf2. Collectively, the current study shows that Nrf2 promotes hepatoma cell growth and progression, partly through induction of PGD transcription.

Resveratrol suppresses gastric cancer cell proliferation and survival through inhibition of PIM-1 kinase activity

The proviral integration site for Moloney murine leukemia virus (PIM) family of serine/threonine-specific kinases consist of three isoforms, that regulate proliferation, apoptosis, metabolism, invasion, and metastasis of cancer cells. Among these, abnormally elevated kinase activity of PIM-1 contributes to the progression of gastric cancer and predicts poor prognosis and a low survival rate in gastric cancer patients. In the present study, we found that resveratrol, one of the representative chemopreventive and anticarcinogenic phytochemicals, directly binds to PIM-1 and thereby inhibits its catalytic activity in human gastric cancer SNU-601 cells. This resulted in suppression of phosphorylation of the proapoptotic Bad, a known substrate of PIM-1. Resveratrol, by inactivating PIM-1, also inhibited anchorage-independent growth and proliferation of SNU-601 cells. To understand the molecular interaction between resveratrol and PIM-1, we conducted docking simulation and found that resveratrol directly binds to the PIM-1 at the ATP-binding pocket. In conclusion, the proapototic and anti-proliferative effects of resveratrol in gastric cancer cells are likely to be mediated through suppression of PIM-1 kinase activity, which may represent a novel mechanism underlying its chemopreventive and anticarcinogenic actions.

Breast cancer cell debris diminishes therapeutic efficacy through heme oxygenase-1-mediated inactivation of M1-like tumor-associated macrophages

Chemotherapy is commonly used as a major therapeutic option for breast cancer treatment, but its efficacy is often diminished by disruption of patient's anti-tumor immunity. Chemotherapy-generated tumor cell debris could hijack accumulated tumor-associated macrophages (TAMs), provoking tumor recurrence. Therefore, reprogramming TAMs to acquire an immunocompetent phenotype is a promising strategy to potentiate therapeutic efficacy. In this study, we analyzed the proportion of immune cells in the breast cancer patients who received chemotherapy. To validate our findings in vivo, we used a syngeneic murine breast cancer (4T1) model. Chemotherapy generates an immunosuppressive tumor microenvironment in breast cancer. Here, we show that phagocytic engulfment of tumor cell debris by TAMs reduces chemotherapeutic efficacy in a 4T1 breast cancer model. Specifically, the engulfment of tumor cell debris by macrophages reduced M1-like polarization through heme oxygenase-1 (HO-1) upregulation. Conversely, genetic or pharmacologic inhibition of HO-1 in TAMs restored the M1-like polarization. Our results demonstrate that tumor cell debris-induced HO-1 expression in macrophages regulates their polarization. Inhibition of HO-1 overexpression in TAMs may provoke a robust anti-tumor immune response, thereby potentiating the efficacy of chemotherapy.

Modulation of tumor microenvironment by chemopreventive natural products

The tumor microenvironment provides a niche in which cancer cells and their surrounding stromal cells reside and in which their interactions occur. The cross talk between cancer and stromal cells in the tumor microenvironment promotes many biological processes to support cancer cell growth, invasion, angiogenesis, and metastasis. Recently, not only cancer cells but also multiple types of surrounding stromal cells, including endothelial cells, immune cells, and fibroblasts in the tumor microenvironment, have been recognized to be attractive targets for reducing resistance to anticancer therapy and tumor recurrence. Many natural products present in fruits, vegetables, herbs, spices, and some marine organisms have been reported to inhibit, delay, or reverse multistage carcinogenesis and to inhibit the proliferation of cancerous cells and the self-renewal capacity of preexisting cancer stem-like cells. Some of these naturally occurring chemopreventive and anticarcinogenic substances can modulate the signal transduction involved in maintaining the activities/functions of stromal cells and their interactions with cancer cells within the tumor microenvironment

Resveratrol suppresses 4-hydroxyestradiol-induced transformation of human breast epithelial cells by blocking I kappa B kinase beta-NF-kappa B signalling

Excess estrogen stimulates the proliferation of mammary epithelial cells and hence represents a major risk factor for breast cancer. Estrogen is subjected to cytochrome P450-catalysed oxidative metabolism to produce an oncogenic catechol estrogen, 4-hydroxyestradiol (4-OHE2). 4-OHE2 undergoes redox cycling during which reactive oxygen species (ROS) as well as the chemically reactive estrogen semiquinone and quinone intermediates are produced, thereby contributing to hormonal carcinogenesis. Resveratrol (3,4',5-trihydroxy stilbene), a phytoalexin present in grapes, has been reported to possess chemopreventive and chemotherapeutic activities. In the present study, we examined the inhibitory effects of resveratrol on 4-OHE2-induced transformation of human breast epithelial MCF-10A cells. Resveratrol inhibited migration and anchorage-independent growth of MCF-10A cells treated with 4-OHE2. Resveratrol treatment suppressed the 4-OHE2-induced activation of I kappa B kinase beta (IKK beta) and phosphorylation of I kappa B alpha, and consequently NF-kappa B DNA binding activity and cyclooxygenase-2 (COX-2) expression. Resveratrol suppressed ROS production and phosphorylation of Akt and ERK induced by 4-OHE2 treatment. In conclusion, resveratrol blocks activation of IKK beta-NF-kappa B signalling and induction of COX-2 expression in 4-OHE2-treated MCF-10A cells, thereby suppressing migration and transformation of these cells

4-Hydroxyestradiol induces mammary epithelial cell transformation through Nrf2-mediated heme oxygenase-1 overexpression

Estrogen (17 beta-estradiol, E-2) undergoes oxidative metabolism by CYP1B1 to form 4-hydroxyestradiol (4-OHE2), a putative carcinogenic metabolite of estrogen. Our previous study showed that 4-OHE2-induced production of reactive oxygen species contributed to neoplastic transformation of human breast epithelial (MCF-10A) cells. In this study, 4-OHE2, but not E-2, increased the expression of heme oxygenase-1 (HO-1), a sensor and regulator of oxidative stress, in MCF-10A cells. Silencing the HO-1 gene in MCF-10A cells suppressed 4-OHE2-induced cell proliferation and transformation. In addition, subcutaneous administration of 4-OHE2 markedly enhanced the growth of the MDA-MB-231 human breast cancer xenografts, which was retarded by zinc protoporphyrin, a pharmacological inhibitor of HO-1. 4-OHE2-induced HO-1 expression was mediated by NF-E2-related factor 2 (Nrf2). We speculate that an electrophilic quinone formed as a consequence of oxidation of 4-OHE2 binds directly to Kelch-like ECH-associated protein 1 (Keap1), an inhibitory protein that sequesters Nrf2 in the cytoplasm. This will diminish association between Nrf2 and Keap1. 4-OHE2 failed to interrupt the interaction between Keap1 and Nrf2 and to induce HO-1 expression in Keap1-C273S or C288S mutant cells. Lano-LC-ESI-MS/ MS analysis in MCF-10AKeap1- WT cells which were treated with 4-OHE2 revealed that the peptide fragment containing Cys288 gained a molecular mass of 287.15 Da, equivalent to the addition of a single molecule of 4-OHE2-derived ortho-quinones

Gremlin-1 Promotes Metastasis of Breast Cancer Cells by Activating STAT3-MMP13 Signaling Pathway

Gremlin-1 (GREM1), one of the bone morphogenetic protein (BMP) antagonists, can directly bind to BMPs. GREM1 is involved in organogenesis, tissue differentiation, and organ fibrosis. Recently, numerous studies have reported the oncogenic role of GREM1 in cancer. However, the role of GREM1 in metastasis of breast cancer cells and its underlying mechanisms remain poorly understood. The role of GREM1 in breast cancer progression was assessed by measuring growth, migration, and invasion of breast cancer cells. An orthotopic breast cancer mouse model was used to investigate the role of GREM1 in lung metastasis of breast cancer cells. GREM1 knockdown suppressed the proliferation of breast cancer cells, while its overexpression increased their growth, migration, and invasion. Cells with Grem1-knockdown showed much lower tumor growth rates and lung metastasis than control cells. GREM1 enhanced the expression of matrix metalloproteinase 13 (MMP13). A positive correlation between GREM1 and MMP13 expression was observed in breast cancer patients. GREM1 activated signal transducer and activator of transcription 3 (STAT3) transcription factor involved in the expression of MMP13. Our study suggests that GREM1 can promote lung metastasis of breast cancer cells through the STAT3-MMP13 pathway. In addition, GREM1 might be a promising therapeutic target for breast cancer metastasis.

KG-135 inhibits COX-2 expression by blocking the activation of JNK and AP-1 in phorbol ester-stimulated human breast epithelial cells

Ginsenosides, ingredients of ginseng, have a wide array of pharmacologic effects. Especially, ginsenosides Rk1, Rg3, and Rg5 derived from heat-processed ginseng have been shown to possess substantial anti-tumor-promoting effects. KG-135 is a formulated complex that contains several antitumorigenic ginsenosides, such as Rk1, Rg3, Rg5, Rk2, Rk3, Rs3, Rs4, Rs5, Rs6, Rs7, etc. The present article was aimed at evaluating the chemopreventive as well as anti-inflammatory effects of KG-135 in the human breast epithelial cell line (MCF-10A). One of the well-recognized molecular targets for chemoprevention is cyclooxygenase-2 (COX-2) that is abnormally upregulated in many premalignant and malignant tissues and cells. In this study, we found that KG-135 inhibited COX-2 expression in MCF-10A cells stimulated with a prototype tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA). Since the transcription factor activator protein-1 (AP-1) plays a role in tumor promotion and is also known to regulate COX-2 induction, we attempted to determine the effect of KG-135 on TPA-induced activation of AP-1. Cotreatment with KG-135 resulted in a decrease in TPA-induced DNA binding of AP-1. In addition, KG-135 inhibited TPA-induced phosphorylation of c-Jun N-terminal kinases (JNK) that regulates COX-2 expression in MCF-10A cells. The JNK inhibitor SP600125 attenuated COX-2 expression in TPA-treated MCF-10A cells. Taken together, the above findings suggest that KG-135 inhibits TPA-induced COX-2 expression in MCF-10A cells by blocking the JNK/AP-1 signaling pathway

Docosahexaenoic acid induces expression of heme oxygenase-1 and NAD(P)H: Quinone oxidoreductase through activation of Nrf2 in human mammary epithelial cells

Docosahexaenoic acid (DHA), an omega-3 fatty acid abundant in fish oils, has diverse health beneficial effects, such as anti-oxidative, anti-inflammatory, neuroprotective, and chemopreventive activities. In this study, we found that DHA induced expression of two representative antioxidant/ cytoprotective enzymes, heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase (NQO1), in human mammary epithealial (MCF-10A) cells. DHA-induced upregulation of these enzymes was accompanied by enhanced translocation of the redox-sensitive transcription factor Nrf2 into the nucleus and its binding to antioxidant response element. Nrf2 gene silencing by siRNA abolished the DHA-induced expression of HO-1 and NQO1 proteins. When MCF-10A cells were transfected with mutant constructs in which the cysteine 151 or 288 residue of Keap1 was replaced by serine, DHA-induced expression of HO-1 and NQO1 was markedly reduced. Moreover, DHA activated protein kinase C (PKC)delta and induced Nrf2 phosphorylation. DHA-induced phosphorylation of Nrf2 was abrogated by the pharmacological PKC delta inhibitor rottlerin or siRNA knockdown of its gene expression. The antioxidants N-acetyl-L-cysteine and Trolox attenuated DHA-induced activation of PKC delta, phosphorylation of Nrf2, and and its target protein expression. In conclusion, DHA activates Nrf2, possibly through modification of critical Keap1 cysteine 288 residue and PKC delta-mediated phosphorylation of Nrf2, leading to upregulation of HO-1 and NQO1 expression