Tamoxifen resistance and metastasis of human breast cancer cells were mediated by the membrane-associated estrogen receptor ER-α36 signaling in vitro

The drug resistance and tumor metastasis have been the main obstacles for the longer-term therapeutic effects of tamoxifen (TAM) on estrogen receptor-positive (ER⁺) breast cancer, but the mechanisms underlying the TAM resistance are still unclear. Here, we demonstrated that the membrane-associated estrogen receptor ER-α36 signaling, but not the G protein-coupled estrogen receptor 1 (GPER1) signaling, might be involved in the TAM resistance and metastasis of breast cancer cells. In this study, a model of ER⁺ breast cancer cell MCF-7 that involves the up-regulated expression of ER-α36 and unchanged expression of ER-α66 and GPER1 was established via the removal of insulin from the cell culture medium. The mechanism of TAM resistance in the ER⁺ breast cancer cell line MCF-7 was investigated, and the results showed that the stimulating effect of insulin on susceptibility of MCF-7 to TAM was mediated by ER-α36 and that the expression level of ER-α36 in TAM-resistant MCF-7 cells was also significantly increased. Both TAM and estradiol (E2) could promote the migration of triple negative (ER-α66^?/PR^?/HER2^?) and ER-α36⁺/GPER1⁺ breast cancer cells MDA-MB-231. The migration of MDA-MB-231 cells was inhibited by the down-regulated intracellular expression of ER-α36 by transient transfection of specific small interfering RNA, whereas no effect of GPER1 down-regulation was observed. Meanwhile, the effect of TAM on the migration of ER-α36-down-regulated MDA-MB-231 cells was also reduced. Furthermore, it was found that TAM enhanced the distribution of integrin β1 on the cell surface but did not affect the expression of integrin β1 in MDA-MB-231 cells. Collectively, these data suggested that ER-α36 signaling might play critical roles in acquired and de novo TAM resistance and metastasis of breast cancer, and ER-α36 might present a potential biomarker of TAM resistance in the clinical diagnosis and treatment of ER⁺ breast cancer.     

Zinc downregulates HIF-1α and inhibits its activity in tumor cells in vitro and in vivo

Background

Hypoxia inducible factor-1α (HIF-1α) is responsible for the majority of HIF-1-induced gene expression changes under hypoxia and for the “angiogenic switch” during tumor progression. HIF-1α is often upregulated in tumors leading to more aggressive tumor growth and chemoresistance, therefore representing an important target for antitumor intervention. We previously reported that zinc downregulated HIF-1α levels. Here, we evaluated the molecular mechanisms of zinc-induced HIF-1α downregulation and whether zinc affected HIF-1α also in vivo.

Methodology/Principal Findings

Here we report that zinc downregulated HIF-1α protein levels in human prostate cancer and glioblastoma cells under hypoxia, whether induced or constitutive. Investigations into the molecular mechanisms showed that zinc induced HIF-1α proteasomal degradation that was prevented by treatment with proteasomal inhibitor MG132. HIF-1α downregulation induced by zinc was ineffective in human RCC4 VHL-null renal carcinoma cell line; likewise, the HIF-1αP402/P564A mutant was resistant to zinc treatment. Similarly to HIF-1α, zinc downregulated also hypoxia-induced HIF-2α whereas the HIF-1β subunit remained unchanged. Zinc inhibited HIF-1α recruitment onto VEGF promoter and the zinc-induced suppression of HIF-1-dependent activation of VEGF correlated with reduction of glioblastoma and prostate cancer cell invasiveness in vitro. Finally, zinc administration downregulated HIF-1α levels in vivo, by bioluminescence imaging, and suppressed intratumoral VEGF expression.

Conclusions/Significance

These findings, by demonstrating that zinc induces HIF-1α proteasomal degradation, indicate that zinc could be useful as an inhibitor of HIF-1α in human tumors to repress important pathways involved in tumor progression, such as those induced by VEGF, MDR1, and Bcl2 target genes, and hopefully potentiate the anticancer therapies.     

JWA reverses cisplatin resistance via the CK2—XRCC1 pathway in human gastric cancer cells

Gastric cancer is the third most common malignancy in China, with a median 5-year survival of only 20%. Cisplatin has been used in first-line cancer treatment for several types of cancer including gastric cancer. However, patients are often primary resistant or develop acquired resistance resulting in relapse of the cancer and reduced survival. Recently, we demonstrated that the reduced expression of base excision repair protein XRCC1 and its upstream regulator JWA in gastric cancerous tissues correlated with a significant survival benefit of adjuvant first-line platinum-based chemotherapy as well as XRCC1 playing an important role in the DNA repair of cisplatin-resistant gastric cancer cells. In the present study, we demonstrated the role of JWA in cisplatin-induced DNA lesions and aquired cisplatin resistance in five cell-culture models: gastric epithelial cells GES-1, cisplatin-sensitive gastric cancer cell lines BGC823 and SGC7901, and the cisplatin-resistant gastric cancer cell lines BGC823/DDP and SGC7901/DDP. Our results indicated that JWA is required for DNA repair following cisplatin-induced double-strand breaks (DSBs) via XRCC1 in normal gastric epithelial cells. However, in gastric cancer cells, JWA enhanced cisplatin-induced cell death through regulation of DNA damage-induced apoptosis. The protein expression of JWA was significantly decreased in cisplatin-resistant cells and contributed to cisplatin resistance. Interestingly, as JWA upregulated XRCC1 expression in normal cells, JWA downregulated XRCC1 expression through promoting the degradation of XRCC1 in cisplatin-resistant gastric cancer cells. Furthermore, the negative regulation of JWA to XRCC1 was blocked due to the mutation of 518S/519T/523T residues of XRCC1, and indicating that the CK2 activated 518S/519T/523T phosphorylation is a key point in the regulation of JWA to XRCC1. In conclusion, we report for the first time that JWA regulated cisplatin-induced DNA damage and apoptosis through the CK2—P-XRCC1—XRCC1 pathway, indicating a putative drug target for reversing cisplatin resistance in gastric cancer.Gastric cancer (GC) is the fifth most common human malignant tumor worldwide but third cause of cancer death.¹ In 2012, there were 405 000 new GC cases diagnosed and 325 000 deaths in China.¹ Current strategy for treatment of GC includes surgery with chemotherapy for potentially curable disease and chemotherapy only for advanced disease. Unfortunately, owing to intrinsic or acquired drug resistance, relapse and metastasis are common and result in high mortality of GC.²Cisplatin is a widely used chemotherapeutic drug for treating various tumors including GC.³ Cisplatin triggers apoptosis by inducing DNA damage through crosslinking of the DNA.⁴ However, cancer cells often develop multiple mechanisms to overcome cisplatin-induced DNA damage and apoptosis, and lead to cisplatin resistance.^{5, 6} Two of the major systems activated are enhanced capability of DNA repair and anti-apoptosis signaling pathways.^{7, 8}XRCC1 is a key mediator of single-strand break DNA repair, and is involved in the process of cisplatin-induced DNA damage repair in various tumors.^{9, 10, 11} XRCC1 was found to identify and bind to DNA interstrand crosslinks induced by cisplatin.¹² Moreover casein kinase 2 (CK2) phosphorylates XRCC1 and is required for its stability and efficient DNA repair.¹³ A selective small molecule inhibitor of CK2, CX-4945, was found to block the cisplatin-induced DNA repair response by decreasing the phosphorylation of XRCC1 at CK2-specific phosphorylation sites.¹⁴ This body of evidence indicates a critical role of XRCC1 and CK2 in cisplatin resistance.The JWA gene, also known as ARL6ip5, was initially cloned from human tracheal bronchial epithelial cells after treatment with all-trans retinoic acid.¹⁵ Subsequent studies indicated that JWA is involved in the cellular responses to heat shock and chemical-mediated oxidative stresses.^{16, 17} Moreover, JWA functions as a base excision repair protein in oxidative-stress-induced DNA single-strand breaks in NIH-3T3 and HELF cells, as evidenced by the positive regulation of XRCC1 levels through MAPK signal pathway and protecting XRCC1 protein from ubiquitination and degradation by proteasome.^{18, 19} However, JWA is also a structurally novel microtubule-binding protein, which regulates cancer cell migration via MAPK cascades and mediates differentiation of leukemic cells.^{20, 21, 22} JWA significantly inhibits melanoma adhesion, invasion and metastasis via integrin aVb3 signaling.²³ More recent data have shown that JWA is required for As₂O₃-induced apoptosis in HeLa and MCF-7 cells via reactive oxygen species and mitochondria-linked signal pathway or promoted p38 MAPK-linked tubulin polymerization.^{24, 25} These reports indicate that the JWA functions as a tumor suppressor for tumor initiation and development.Recently, we reported the prognostic and predictive role of JWA and XRCC1 expression in GC. JWA and XRCC1 protein levels are significantly downregulated in GC lesions compared with adjacent noncancerous tissues, whereas platinum-based chemotherapy significantly improved overall survival in GC patients with low levels of tumoral JWA or XRCC1 expression.²⁶ Subsequent studies indicated that overexpression of XRCC1 contributed to cisplatin resistance in GC cells and showed that XRCC1 protein was important for effective repair of cisplatin-induced DSBs in GC cells.²⁷ However, the contribution of JWA to cisplatin resistance in GC and underlying mechanisms are not fully understood.The objectives of the present study were to investigate the role of JWA in cisplatin resistance of GC cells and elucidate the underlying mechanisms of action. Our results demonstrated that JWA negatively regulated XRCC1 through the CK2—p-XRCC1 pathway in cisplatin-resistant GC cells. The JWA could be a valuable target for reversal of cisplatin resistance in human GC.     

Downregulation of fatty acid oxidation by involvement of HIF-1α and PPARγ in human gastric adenocarcinoma and related clinical significance

Journal of Physiology and Biochemistry - Lipid metabolism rewiring in gastric adenocarcinoma (GA) pathogenesis is still not clearly elucidated. This study aimed to describe the role of lipid...     

Interleukin-23 is constitutively expressed in the human annulus in vivo and in vitro,and is up-regulated in vitro by TNF-α

ABSTRACT

Interleukin-23 (IL-23, IL-23p19) is a proinflammatory cytokine in the IL-12-related family. Although inflammatory cells in herniated discs have been shown to contain IL-23, little is known about the presence and role of IL-23 in human disc cells. We analyzed disc specimens for IL-23 localization using immunohistochemistry in control, herniated and non-herniated discs from which annulus fibrosus (annulus) cells were isolated and cultured to identify IL-23 gene expression and production. Microarray analysis was used to assess the expression of IL-23 in disc tissue and in cells exposed to two proinflammatory cytokines, IL-1ß and TNF-α. IL-23 was present in annulus cells at the protein level and its expression was up-regulated significantly in herniated compared to control disc tissue. Direct measurement of medium components confirmed production of IL-23 and its receptor, IL-23R, by annulus cells in vitro. Annulus cells in three-dimensional culture exposed to TNF-α, but not IL-1ß, resulted in significant up-regulation of IL-23 expression compared to control cells. Our findings are evidence for the constitutive presence of IL-23 in the human disc and that its expression in vitro is modified by exposure to TNF-α.     

HIF-1α signaling is augmented during intermittent hypoxia by induction of the Nrf2 pathway in NOX1-expressing adenocarcinoma A549 cells

Fluctuations in cellular oxygenation causing intermittent hypoxia and oxidative stress affect the regulation of hypoxia-inducible factor (HIF-1) and the nuclear factor erythroid 2-related factor 2 (Nrf2). HIF-1 is primarily induced in hypoxia, whereas Nrf2 is induced in response to oxidative stress. Whereas HIF-1 regulates the expression of genes important for the adaptation of cells to hypoxia, Nrf2 induces antioxidative enzymes such as thioredoxin 1 (Trx1), exerting a cytoprotective role. Here, we investigated the regulation and cross talk of HIF-1α and Nrf2 in intermittent hypoxia in lung adenocarcinoma A549 cells expressing high levels of the NADPH oxidase subunit NOX1. Whereas continuous hypoxia induced only HIF-1α, intermittent hypoxia induced both HIF-1α and Nrf2, including its target Trx1. NOX1 was determined to be crucial for enhanced ROS production in intermittent hypoxia that in turn mediated induction of Nrf2 and Trx1. The regulation of Nrf2 and Trx1 by NOX1 was confirmed by both inhibition of endogenous NOX1 and overexpression of recombinant NOX1 protein. By using a proteasomal inhibitor, NOX1 was demonstrated to activate Nrf2 by protein stabilization. Subsequently, Nrf2-dependent Trx1 induction turned out to enhance HIF-1α signaling in intermittent hypoxia.     

Dehydrocostuslactone suppresses angiogenesis in vitro and in vivo through inhibition of Akt/GSK-3β and mTOR signaling pathways

The traditional Chinese medicine component dehydrocostuslactone (DHC) isolated from Saussurea costus (Falc.) Lipschitz, has been shown to have anti-cancer activity. Angiogenesis is an essential process in the growth and progression of cancer. In this study, we demonstrated, for the first time, the anti-angiogenic mechanism of action of DHC to be via the induction of cell cycle progression at the G0/G1 phase due to abrogation of the Akt/glycogen synthase kinase-3β (GSK-3β)/cyclin D1 and mTOR signaling pathway. First, we demonstrated that DHC has an anti-angiogenic effect in the matrigel-plug nude mice model and an inhibitory effect on human umbilical vein endothelial cell (HUVEC) proliferation and capillary-like tube formation in vitro. DHC caused G0/G1 cell cycle arrest, which was associated with the down-regulation of cyclin D1 expression, leading to the suppression of retinoblastoma protein phosphorylation and subsequent inhibition of cyclin A and cdk2 expression. With respect to the molecular mechanisms underlying the DHC-induced cyclin D1 down-regulation, this study demonstrated that DHC significantly inhibits Akt expression, resulting in the suppression of GSK-3β phosphorylation and mTOR expression. These effects are capable of regulating cyclin D1 degradation, but they were significantly reversed by constitutively active myristoylated (myr)-Akt. Furthermore, the abrogation of tube formation induced by DHC was also reversed by overexpression of Akt. And the co-treatment with LiCl and DHC significantly reversed the growth inhibition induced by DHC. Taken together, our study has identified Akt/GSK-3β and mTOR as important targets of DHC and has thus highlighted its potential application in angiogenesis-related diseases, such as cancer.     

Docosahexaenoic acid inhibited the Wnt/β-Catenin pathway and suppressed breast cancer cells in vitro and in vivo

N-3 fatty acids (FAs) are essential FAs necessary for human health and are known to possess anticancer properties. However, the relationship between n-3 FAs and β-catenin, one of the key components of the Wnt signaling pathway, in mouse breast cancer remains poorly characterized. In this study, 4T1 mouse breast cancer cells were exposed to a representative n-3 FA, docosahexaenoic acid (DHA), to investigate the relationship between n-3 FAs and the Wnt/β-catenin signaling pathway in vivo and in vitro. In vitro studies showed that DHA strongly inhibited cell growth, and induced G1 cell cycle arrest both in 4T1 mouse breast cells and MCF-7 human breast cells. DHA reduced β-catenin expression and T cell factor/lymphoid-enhancing factor reporter activity in 4T1 mouse breast cells. In addition, DHA down-regulated the expression of downstream target genes such as c-myc and cyclinD1. In vivo, therapy experiments were conducted on Babl/c mice bearing breast cancer. We found that feeding mouse the 5% fish oil-supplemented diet for 30 days significantly reduced the growth of 4T1 mouse breast cancer in vivo through inhibition of cancer cell proliferation as well as induction of apoptosis. Feeding animals a 5% fish oil diet significantly induced down-regulation of β-catenin in tumor tissues with a notable increase in apoptosis. In addition, fish oil-supplemented diet decreased lung metastases of breast cancer. These observations suggested that DHA exerted its anticancer activity through down-regulation of Wnt/β-catenin signaling. Thus, our data call for further studies to assess the effectiveness of fish oil as a dietary supplement in the prevention and treatment of breast cancer.     

MARK2/4 promotes Warburg effect and cell growth in non-small cell lung carcinoma through the AMPKα1/mTOR/HIF-1α signaling pathway

MARKs kinase belongs to an AMPK-related family kinase plays a critical role in tumor progression, but its exact role and contribution of four different isoforms remain largely ambiguous. In this study, we used a clinical dataset compiled by The Cancer Genome Atlas (TCGA) and GEO revealed that MARK2 and MARK4 expressions were significantly upregulated in non-small cell lung cancer (NSCLC) compared with normal tissues. Furthermore, expressions of MARK2/4 were highly appeared in advanced stages and associated with the low survival rate of NSCLC patients. Functional assays demonstrated that MARK2/4 deletion or MARKs inhibition significantly suppressed aerobic glycolysis and cell growth in NSCLC cells. Mechanistically, MARK2/4 stimulates the mTOR/HIF-1α pathway and subsequently alleviates AMPK activity via physically associate with Raptor and AMPKα1, thereby facilitating aerobic glycolysis and cell growth in NSCLC cells. However, these effects were markedly reversed by MARKs inhibitor 39621, or MARK2/4 deletion, mTOR inhibitor rapamycin, or AMPK activator AICAR. Together, the data demonstrated that MARK2/4 exerts its oncogenic effects by facilitating metabolic reprogramming in NSCLC cells. Therefore, MARK2/4 might be a potential therapeutic target for lung cancer.     

Antitumor effects of human recombinant interleukin-1α and etoposide against human tumor cells: mechanism for synergismin vitro and activityin vivo

Recombinant human interleukin 1α (rh IL-1α) and etoposide (VP-16) synergize for direct growth inhibition of several human tumor cell linesin vitro. Our previous studies demonstrated that VP-16 increased the number of membrane-associated IL-1 receptors (IL-1Rs) and also enhanced the internalization of receptor-bound rh IL-1α. The purposes of this study were to test our hypothess that these events were critical to the synergy between rhIL-1α and VP-16, to determine whether rhIL-1α and VP-16 synergize to increase superoxide (SO) anion radical productionin vitro since SO anion has been implicated in the toxic effects of IL-1, and to investigate the antitumor efficacy of the combinaton against tumors in vivo. A375/C6 melanoma cells and OVCAR-3 ovarian carcinoma cells were tested with IL-1 receptor antagonist (IL-1ra) before exposure to rhIL-1α, VP-16 and rhIL-1α plus VP-16. The synergistic or antagonistic effects were assessed by MTT assay. SO production was measured by reduction of cytochrome C. Athymic female mice bearing the A375/C6 melanoma were treated by rhIL-1α, VP-16, and rhIL-1α+VP-16. The antitumor effects were evaluated by quantitating tumor growth and survival time. Pretreatment with the IL-1ra abrogated the synergistic effects of rhIL-1α and VP-16. The production of SO radical by A375/C6 cells was increased 2.5 fold by the combination of rhIL-1α and VP-16, and the addition of exogenous SOD blocked the synergy between rhIL-1α and VP-16. However, when A375/S0D15 cells which over-expressed manganese superoxide dismutase (MnSOD) after MnSOD cDNA transfecton were exposed to rhIL-1α and VP-16, in vitro antagonism was observed. In vivo studies demonstrated that the combination of rhIL-1α and VP-16 delayed tumor growth better than either agent alone, although long-term survival was not improved because of substantial toxicity. Our results suggest that the synergistic antitumor effects of IL-1α and VP-16 may be due to IL-1R modulation and increased internalization of IL-1-IL-1R complex by VP-16 treatment, as well as to a subsequent increase in SO anion radical production from the tumor cells exposed to both drugs. Thus, the combnation of IL-1α and VP-16 might prove useful for the treatment of malignant diseasein vivo, if the increased toxicity can be reduced or managed. The US Government’s right to retain a non-exclusive royalty-free license on and to any copyright is acknowledged.     

Wnt/β-catenin signaling is critical for dedifferentiation of aged epidermal cells in vivo and in vitro

Aged epidermal cells have the capacity to dedifferentiate into stem cell-like cells. However, the signals that regulate the dedifferentiation of aged epidermal cells remain unclear. Here, we provide evidence that Wnt/β-catenin is critical for aged epidermal cell dedifferentiation in vivo and in vitro. Some aged epidermal cells in human ultrathin epidermal sheets lacking basal stem cells transplanted onto wounds dedifferentiated into stem cell-like cells that were positive for CK19 and β1 integrin but negative for CK10. In addition, Wnt/β-catenin pathway was activated during this process. There was increased expression of Wnt-1, Wnt-4, Wnt-7a, β-catenin, cyclin D1, and c-myc. Secreted frizzled-related protein 1, a Wnt/β-catenin pathway inhibitor, blocked dedifferentiation in vivo. Then, the activator, a highly specific glycogen synthase kinase (GSK)-3β inhibitor, of Wnt/β-catenin pathway was added to the culture medium of aged epidermal cells. Surprisingly, we found that the activator induced higher expression of CK19, β1 integrin, Oct4, and Nanog proteins. The induced aged epidermal cells exhibited high colony-forming efficiency, long-term proliferative potential and could regenerate a skin equivalent (as do epidermal stem cells). These results suggested that activation of Wnt/β-catenin pathway induced the dedifferentiation of aged epidermal cells, which suggest a new approach to generate epidermal stem cell-like cells.     

Interferon-α/β and anti-fibroblast growth factor receptor 1 monoclonal antibody suppress hepatic cancer cells in vitro and in vivo

Background

Hepatocellular carcinoma (HCC) is the most commonly occurring primary liver cancer and ranks as the fifth most frequently occurring cancer, overall, and the third leading cause of cancer deaths, worldwide. At present, effective therapeutic options available for HCC are limited; consequently, the prognosis for these patients is poor. Our aim in the present study was to identify a novel target for antibody therapy against HCC.

Methodology/Principal Findings

We used Western blot and flow cytometric and immunocytochemical analyses to investigate the regulation of FGFR1 expression by interferon-α/β in several human hepatic cancer cell lines. In addition, we tested the efficacy of combined treatment with anti-FGFR1 monoclonal antibody and interferon-α/β in a murine xenograft model of human HCC. We found that interferon-α/β induces expression of FGFR1 in human HCC cell lines, and that an anti-FGFR1 monoclonal antibody (mAb) targeting of the induced FGFR1 can effectively inhibit growth and survival of HCC cells in vitro and in vivo. Moreover, the combination of interferon-α, anti-FGFR1 mAb and peripheral blood mononuclear cells (PBMCs) exerted a significant antitumor effect in vitro.

Conclusions

Our results suggest that the combined use of an anti-FGFR1 antibody and interferon-α/β is a promising approach to the treatment of HCC.