UCP2 inhibition sensitizes breast cancer cells to therapeutic agents by increasing oxidative stress

Modulation of oxidative stress in cancer cells plays an important role in the study of the resistance to anticancer therapies. Uncoupling protein 2 (UCP2) may play a dual role in cancer, acting as a protective mechanism in normal cells, while its overexpression in cancer cells could confer resistance to chemotherapy and a higher survival through downregulation of ROS production. Thus, our aim was to check whether the inhibition of UCP2 expression and function increases oxidative stress and could render breast cancer cells more sensitive to cisplatin (CDDP) or tamoxifen (TAM). For this purpose, we studied clonogenicity, mitochondrial membrane potential (ΔΨm), cell viability, ROS production, apoptosis, and autophagy in MCF-7 and T47D (only the last four determinations) breast cancer cells treated with CDDP or TAM, in combination or without a UCP2 knockdown (siRNA or genipin). Furthermore, survival curves were performed in order to check the impact of UCP2 expression in breast cancer patients. UCP2 inhibition and cytotoxic treatments produced a decrease in cell viability and clonogenicity, in addition to an increase in ΔΨm, ROS production, apoptosis, and autophagy. It is important to note that CDDP decreased UCP2 protein levels, so that the greatest effects produced by the UCP2 inhibition in combination with a cytotoxic treatment, with regard to treatment alone, were observed in TAM+UCP2siRNA-treated cells. Moreover, this UCP2 inhibition caused autophagic cell death, since apoptosis parameters barely increased after UCP2 knockdown. Finally, survival curves revealed that higher UCP2 expression corresponded with a poorer prognosis. In conclusion, UCP2 could be a therapeutic target in breast cancer, especially in those patients treated with tamoxifen.     

P300 binds to and acetylates MTA2 to promote colorectal cancer cells growth

MTA2 is a member of metastasis associated family, which is highly expressed in several solid tumors and associated with tumor cells migration and invasion. Here, we report that MTA2 is acetylated at K152 and histone acetyltransferase p300 binds to and acetylates MTA2. Furthermore, mutation of the MTA2 acetylation site inhibits the growth of colorectal cancer cells and migration and invasion of Rat1 fibroblasts. These results reveal a novel post-translational regulation of MTA2 by the way of p300-dependent acetylation, which is important for tumor cells growth and migration and provides a potential target for clinical cancer research.     

Three-dimensional culture sensitizes epithelial ovarian cancer cells to EZH2 methyltransferase inhibition

Inhibitors of EZH2 methyltransferase activity have been demonstrated to selectively suppress the growth of diffused large B cell lymphoma (DLBCL) cells with gain-of-function mutations in EZH2, while exhibiting very limited effects on the growth of DLBCL cells with wild-type EZH2. Given that EZH2 is often overexpressed but not mutated in solid tumors, it is important to investigate the determinants of sensitivity of solid tumor cells to EZH2 inhibitors. In the current study, we show that three-dimensional (3D) culture of epithelial ovarian cancer (EOC) cells that overexpress EZH2 sensitizes these cells to EZH2 methyltransferase inhibition. Treatment of EOC cells with GSK343, a specific inhibitor of EZH2 methyltransferase, decreases the level of H3K27Me3, the product of EZH2’s enzymatic activity. However, GSK343 exhibited limited effects on the growth of EOC cells in conventional two-dimensional (2D) culture. In contrast, GSK343 significantly suppressed the growth of EOC cells cultured in 3D matrigel extracellular matrix (ECM), which more closely mimics the tumor microenvironment in vivo. Notably, GSK343 induces apoptosis of EOC cells in 3D but not 2D culture. In addition, GSK343 significantly inhibited the invasion of EOC cells. In summary, we show that the 3D ECM sensitizes EOC cells to EZH2 methyltransferase inhibition, which suppresses cell growth, induces apoptosis and inhibits invasion. Our findings imply that in EZH2 wild-type solid tumors, the ECM tumor microenvironment plays an important role in determining sensitivity to EZH2 inhibition and suggest that targeting the ECM represents a novel strategy for enhancing EZH2 inhibitor efficacy.     

Inhibition of PARP1 activity enhances chemotherapeutic efficiency in cisplatin-resistant gastric cancer cells

Cisplatin (DDP) is the first line chemotherapeutic drug for several cancers, including gastric cancer (GC). Unfortunately, the rapid development of drug resistance remains a significant challenge for the clinical application of cisplatin. There is an urgent need to develop new strategies to overcome DDP resistance for cancer treatment. In this study, four types of human GC cells have been divided into naturally sensitive or naturally resistant categories according to their responses to cisplatin. PARP1 activity (poly (ADP-ribose), PAR) was found to be greatly increased in cisplatin-resistant GC cells. PARP1 inhibitors significantly enhanced cisplatin-induced DNA damage and apoptosis in the resistant GC cells via the inhibition of PAR. Mechanistically, PARP1 inhibitors suppress DNA-PKcs stability and reduce the capability of DNA double-strand break (DSB) repair via the NHEJ pathway. This was also verified in BGC823/DDP GC cells with acquired cisplatin resistance. In conclusion, we identified that PARP1 is a useful interceptive target in cisplatin-resistant GC cells. Our data provide a promising therapeutic strategy against cisplatin resistance in GC cells that has potential translational significance.     

Depression of MAD2 inhibits apoptosis of gastric cancer cells by upregulating Bcl-2 and interfering mitochondrion pathway

Mitotic arrest deficient 2 (MAD2) is an essential component of the mitotic spindle checkpoint pathway. It was previously shown to be associated with drug resistance of tumor cells. To further explore the roles of MAD2 in responses of gastric cancer cells to chemotherapy drugs, we constructed the siRNA vectors of MAD2 and transfected them into gastric cancer SGC7901 cells to inhibit expression of MAD2. MTT assay showed that the downregulation of MAD2 increased the resistance of SGC7901 cells to spindle inhibitors and DNA damaging agents. The apoptosis rates of gastric cancer cells transfected with MAD2-siRNA were 10.7% and 10%, respectively, after treated by 1.0microg/ml VCR and cisplatin. In contrast, the apoptosis rates of SGC7901 and SGC7901/psilencer3.1 induced by VCR were 43.2%, 38.7%; and that induced by cispaltin were 34.1%, 31.4%. The ratio of Bcl-2 to Bax was much higher in the MAD2-siRNA transfectants compared with the SGC7901/psilencer. In SGC7901/psilencer, cytochrome c and cleaved caspase 3 protein levels increased along with the exposure time increased. However, these protein levels of SGC7901/MAD2-siRNA had no changes during the drug treatment. These results indicate that down regulation of MAD2 could promote the drug resistance of gastric cancer cells and inhibit anticancer drugs induced-apoptosis by upregulating Bcl-2 and interfering the mitochondrion apoptosis pathway.     

Valproic acid sensitizes breast cancer cells to hydroxyurea through inhibiting RPA2 hyperphosphorylation-mediated DNA repair pathway

It was reported that valproic acid (VPA, a histone deacetylase inhibitor) can sensitize cancer cells to hydroxyurea (HU, a ribonucleotide reductase inhibitor) for chemotherapy, although the mechanism of VPA-induced HU sensitization is unclear. In this study, we systematically characterized VPA-induced HU sensitization of breast cancer cells. Multiple breast cancer cell models were employed to investigate whether the safe concentration of 0.5 mM VPA and 2 mM HU can result in DNA double-strand breaks (DSBs) and impact cell survival. Furthermore, the underlying mechanism was explored through cell biology assays, including clonogenic survival, homologous recombination (HR) activity, immunoblot and immunofluorescence. We found that VPA and HU cooperatively suppressed cancer cell survival. VPA resulted in the accumulation of more DNA double-strand breaks (DSBs) in response to HU-induced replication arrest and was able to block HU-stimulated homologous recombination (HR) through inhibiting the activity of two key HR repair proteins by hyperphosphorylation of replication protein A2 (RPA2-p) and recombinase Rad51. However, apoptosis was not detected under this condition. In addition, the results from the survival fraction in the cells expressing defective RPA2-p showed that VPA disrupted the HU-induced RPA2-p-Rad51-mediated HR pathway. Importantly, these findings were further supported by analyzing primary-culture cells from the tissue of chemical carcinogen (DMBA)-induced breast cancer in rats. Thus, our data demonstrated that VPA and HU synergistically suppressed tumor cells via disturbing RPA2-p-mediated DNA repair pathway, which provides a new way for combining chemotherapeutic drugs to sensitize breast cancer cells.     

Cellular responses to replication stress: Implications in cancer biology and therapy

DNA replication is essential for cell proliferation. Any obstacles during replication cause replication stress, which may lead to genomic instability and cancer formation. In this review, we summarize the physiological DNA replication process and the normal cellular response to replication stress. We also outline specialized therapies in clinical trials based on current knowledge and future perspectives in the field.     

Dampening HOTAIR sensitizes the gastric cancer cells to oxaliplatin through miR-195-5p and ABCG2 pathway

Long non-coding RNAs (lncRNA) have an extensive role in the progression and chemoresistance of gastric cancer (GC). Deeply study the regulatory role of lncRNAs could provide potential therapeutic targets. The aim of this study is to explore the regulatory role of HOTAIR in the progression and oxaliplatin resistance of GC. The expression of HOTAIR in GC and cell lines were detected by using qRT-PCR. Cell proliferation and apoptosis were analysed by CCK-8, EdU incorporation and flow cytometry. Luciferase reporter assay was used to identify the interaction between HOTAIR and ABCG2 (ATP-binding cassette (ABC) superfamily G member 2, ABCG2) via miR-195-5p. The regulatory functions were verified by using molecular biology experiments. HOTAIR was significantly overexpressed in GC and associated with poor prognosis. Knock-down of HOTAIR inhibited the GC cells proliferation and oxaliplatin resistance, while overexpression of HOTAIR showed opposite functions. Further studies found that HOTAIR acted as a competing endogenous RNA (ceRNA) to absorb miR-195-5p and elevated the expression of ABCG2, which leads to resistance of GC cells to oxaliplatin. Taken together, our findings demonstrated that HOTAIR regulates ABCG2 induced resistance of GC to oxaliplatin through miR-195-5p signalling and illustrate the great potential of developing new therapeutic targets for GC patients.     

Inhibition of PKM2 sensitizes triple-negative breast cancer cells to doxorubicin

Cancer cells alter regular metabolic pathways in order to sustain rapid proliferation. One example of metabolic remodeling in cancerous tissue is the upregulation of pyruvate kinase isoenzyme M2 (PKM2), which is involved in aerobic glycolysis. Indeed, PKM2 has previously been identified as a tumor biomarker and as a potential target for cancer therapy. Here, we examined the effects of combined treatment with doxorubicin and anti-PKM2 small interfering RNA (siRNA) on triple-negative breast cancer (TNBC). The suppression of PKM2 resulted in changes in glucose metabolism, leading to decreased synthesis of adenosine triphosphate (ATP). Reduced levels of ATP resulted in the intracellular accumulation of doxorubicin, consequently enhancing the therapeutic efficacy of this drug in several triple-negative breast cancer cell lines. Furthermore, the combined effect of PKM2 siRNA and doxorubicin was evaluated in an in vivo MDA-MB-231 orthotopic breast cancer model. The siRNA was systemically administered through a polyethylenimine (PEI)-based delivery system that has been extensively used. We demonstrate that the combination treatment showed superior anticancer efficacy as compared to doxorubicin alone. These findings suggest that targeting PKM2 can increase the efficacy of chemotherapy, potentially providing a new approach for improving the outcome of chemotherapy in patients with TNBC.     

Methylation of histone H3 at lysine 37 by Set1 and Set2 prevents spurious DNA replication

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Induction of ER stress protects gastric cancer cells against apoptosis induced by cisplatin and doxorubicin through activation of p38 MAPK

We investigated the role of endoplasmic reticulum (ER) stress response and p38 MAPK pathways in the resistance of gastric cancer cells to chemotherapy. Pretreatment of the gastric cancer cells with the ER stress inducer drastically decreased the apoptotic rate induced by cisplatin or doxorubicin. Induction of ER stress also led to the activation of p38. Inhibition of p38 activity abrogated the effects of ER stress-induced resistance to apoptosis induced by cisplatin- and doxorubicin treatment. Thus, ER-stress response in gastric cancer cells causes resistance to cisplatin- and doxorubicin-induced apoptosis, and ER-stress induced chemo-resistance can be overcome by blocking p38 activity.     

MTA1 is a novel regulator of autophagy that induces tamoxifen resistance in breast cancer cells

Tamoxifen is commonly used to treat patients with ESR/ER-positive breast cancer, but its therapeutic benefit is limited by the development of resistance. Recently, alterations in macroautophagy/autophagy function were demonstrated to be a potential mechanism for tamoxifen resistance. Although MTA1 (metastasis-associated 1) has been implicated in breast tumorigenesis and metastasis, its role in endocrine resistance has not been studied. Here, we report that the level of MTA1 expression was upregulated in the tamoxifen resistant breast cancer cell lines MCF7/TAMR and T47D/TR, and knockdown of MTA1 sensitized the cells to 4-hydroxytamoxifen (4OHT). Moreover, knockdown of MTA1 significantly decreased the enhanced autophagy flux in the tamoxifen resistant cell lines. To confirm the role of MTA1 in the development of tamoxifen resistance, we established a cell line, MCF7/MTA1, which stably expressed MTA1. Compared with parental MCF7, MCF7/MTA1 cells were more resistant to 4OHT-induced growth inhibition in vitro and in vivo, and showed increased autophagy flux and higher numbers of autophagosomes. Knockdown of ATG7 or cotreatment with hydroxychloroquine, an autophagy inhibitor, restored sensitivity to 4OHT in both the MCF7/MTA1 and tamoxifen resistant cells. In addition, AMP-activated protein kinase (AMPK) was activated, probably because of an increased AMP:ATP ratio and decreased expression of mitochondrial electron transport complex components. Finally, publicly available breast cancer patient datasets indicate that MTA1 levels correlate with poor prognosis and development of recurrence in patients with breast cancer treated with tamoxifen. Overall, our findings demonstrated that MTA1 induces AMPK activation and subsequent autophagy that could contribute to tamoxifen resistance in breast cancer.     

Celastrol inhibits gastric cancer growth by induction of apoptosis and autophagy

Recently, the interest in natural products for the treatment of cancer is increasing because they are the pre-screened candidates. In the present study, we demonstrate the therapeutic effect of celastrol, a triterpene extracted from the root bark of Chinese medicine on gastric cancer. The proliferation of AGS and YCC-2 cells were most sensitively decreased in six kinds of gastric cancer cell lines after the treatment with celastrol. Celastrol inhibited the cell migration and increased G1 arrest in cell-cycle populations in both cell lines. The treatment with celastrol significantly induced autophagy and apoptosis and increased the expression of autophagy and apoptosis-related proteins. We also found an increase in phosphorylated AMPK following a decrease in all phosphorylated forms of AKT, mTOR and S6K after the treatment with celastrol. Moreover, gastric tumor burdens were reduced in a dose-dependent manner by celastrol administration in a xenografted mice model. Taken together, celastrol distinctly inhibits the gastric cancer cell proliferation and induces autophagy and apoptosis. [BMB Reports 2014; 47(12): 697-702]     

Targeting NPRL2 to enhance the efficacy of Olaparib in castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) lacks effective treatment, and studies have shown that PARPi inhibitors, such as Olaparib, are somewhat effective; however, the efficacy of Olaparib in CRPC still needs to be further improved. Nitrogen permease regulator-like 2 (NPRL2) is reported to be a tumor suppressor candidate gene and is closely related to the DNA repair pathway, which can affect the sensitivity of many chemotherapeutic drugs. However, there is no research on whether NPRL2 is associated with sensitivity to Olaparib. Hence, in the present study, we examined the NPRL2 expression levels in several PCa cell lines (LNCaP, PC3, and enzalutamide-resistant LNCaP, named LNPER) by Western blot. In addition, we investigated the role of NPRL2 expression and silencing in cell proliferation and in the regulation of ataxia telangiectasia mutated (ATM), which can mediate DNA repair and sensitivity to Olaparib. Furthermore, we performed in vitro and in vivo experiments to determine the mechanism of action of NPRL2 in adjusting Olaparib sensitivity. Our findings demonstrated that the NPRL2 expression level was upregulated in PCa cells, especially CRPC cells. NPRL2 overexpression promoted growth and resistance to Olaparib, and NPRL2 silencing inhibited proliferation, enhanced sensitivity to Olaparib, and increased CRRL2 expression in PCa cells. In addition, the Olaparib-induced growth delay in NPRL2-silenced PC3 tumors in mice correlated with ATM signaling downregulation, an apoptosis increase and migration/invasion suppression. Our results indicate that NPRL2 silencing enhances sensitivity to Olaparib treatment in CRPC and that NPRL2 may serve as a potential therapeutic target and predict resistance to Olaparib in CRPC.     

水飞蓟宾抑制TNF-α诱导的MMP-9在胃癌细胞中的表达

目的确定在胃癌细胞株中水飞蓟宾对TNF-α诱导的MMP-9表达的影响。方法应用细胞增殖分析、化学抑制剂处理、免疫印迹、流式细胞分析、腺病毒转移等技术完成实验。结果在SNU216和SNU668胃癌细胞中,MMP-9mRNA和蛋白的表达都被TNF-α剂量依赖性地提高。另-方面,TNF—α诱导的MMP-9表达被水飞蓟宾剂量依赖性地抑制。结论在胃癌细胞中水飞蓟宾可减少TNF-α诱导的MMP-9表达。     

Ubiquitin ligase Cbl-b sensitizes leukemia and gastric cancer cells to anthracyclines by activating the mitochondrial pathway and modulating Akt and ERK survival signals

The present study reported that the ubiquitin ligase Cbl-b was up-regulated during anthracycline-induced apoptosis in two cell lines, RBL-2H3 leukemia cells and MGC803 gastric cancer cells. Overexpression of Cbl-b strongly promoted the cytotoxic and apoptosis-inducing effects of anthracyclines, while a dominant negative (DN) Cbl-b mutation abolished these effects in both cell lines. Further investigation revealed that mitochondrial depolarization was enhanced by Cbl-b and decreased by Cbl-b (DN) in RBL-2H3 cells. Moreover, overexpression of Cbl-b significantly suppressed ERK activation, and Cbl-b (DN) strongly enhanced both ERK and Akt activation. Altogether, these results indicate that Cbl-b sensitized both leukemia and gastric cancer cells to anthracyclines by activating the mitochondrial apoptotic pathway and modulating the ERK and Akt survival pathways.     

Downregulation of PPP2R5E expression by miR-23a suppresses apoptosis to facilitate the growth of gastric cancer cells

PPP2R5E belongs to the phosphatase 2A regulatory subunit B family and acts as a tumor suppressor in human cancer. However, the role of PPP2R5E in the tumorigenesis of gastric cancer is unclear. Here, we declare that PPP2R5E is downregulated by miR-23a and induces cell growth inhibition and apoptosis in gastric cancer cells. Furthermore, ASO-miR-23a suppresses tumor growth derived from MGC803 cells in vivo. PPP2R5E is identified as a new target of miR-23a. Moreover, overexpression of PPP2R5E reversed the negative effects of miR-23a. We highlight the significance of miR-23a and PPP2R5E in the proliferation and apoptosis of gastric cancer cells.     

Proteomics characterization of gastrokine 1-induced growth inhibition of gastric cancer cells

We previously used proteomics technology to globally identify gastric cancer-associated proteins and found that gastrokine 1 (GKN1) was dramatically underexpressed in gastric cancer tissues. Here, we further showed that GKN1 could inhibit cell growth and induce cell cycle arrest in gastric cancer cells. The activity of protein kinase PKCδ/θ was inhibited by GKN1, whereas the activity of ERK1/2 and JNK1/2 was increased by GKN1, suggesting that GKN1 induced growth inhibition of gastric cancer cells by synergistically regulating the activity of these protein kinases. Seventy-four proteins were found to be regulated by GKN1 by proteomics analysis, including α-enolase (ENO1) and Cathepsin D. Interestingly, ENO1 is an important hub in the protein-protein interaction network of the 74 differential proteins. Silencing of ENO1 resulted in growth inhibition and cell cycle arrest of gastric cancer cells, similar to the effect of GKN1 overexpression in cells, whereas ENO1 overexpression blocked GKN1-induced growth inhibition and cell cycle arrest. These observations suggested that ENO1 downregulation played an important role in GKN1-induced growth inhibition of gastric cancer cells.     

Calpain activation through galectin-3 inhibition sensitizes prostate cancer cells to cisplatin treatment

Prostate cancer will develop chemoresistance following a period of chemotherapy. This is due, in part, to the acquisition of antiapoptotic properties by the cancer cells and, therefore, development of novel strategies for treatment is of critical need. Here, we attempt to clarify the role of the antiapoptotic molecule galectin-3 in prostate cancer cells using siRNA and antagonist approaches. The data showed that Gal-3 inhibition by siRNA or its antagonist GCS-100/modified citrus pectin (MCP) increased cisplatin-induced apoptosis of PC3 cells. Recent studies have indicated that cisplatin-induced apoptosis may be mediated by calpain, a calcium-dependent protease, as its activation leads to cleavage of androgen receptor into an androgen-independent isoform in prostate cancer cells. Thus, we examined whether calpain activation is associated with the Gal-3 function of regulating apoptosis. Here, we report that Gal-3 inhibition by siRNA or GCS-100/MCP enhances calpain activation, whereas Gal-3 overexpression inhibits it. Inhibition of calpain using its inhibitor and/or siRNA attenuated the proapoptotic effect of Gal-3 inhibition, suggesting that calpain activation may be a novel mechanism for the proapoptotic effect of Gal-3 inhibition. Thus, a paradigm shift for treating prostate cancer is suggested whereby a combination of a non-toxic anti-Gal-3 drug together with a toxic chemotherapeutic agent could serve as a novel therapeutic modality for chemoresistant prostate cancers.     

Dormant origins as a built-in safeguard in eukaryotic DNA replication against genome instability and disease development

DNA replication is a prerequisite for cell proliferation, yet it can be increasingly challenging for a eukaryotic cell to faithfully duplicate its genome as its size and complexity expands. Dormant origins now emerge as a key component for cells to successfully accomplish such a demanding but essential task. In this perspective, we will first provide an overview of the fundamental processes eukaryotic cells have developed to regulate origin licensing and firing. With a special focus on mammalian systems, we will then highlight the role of dormant origins in preventing replication-associated genome instability and their functional interplay with proteins involved in the DNA damage repair response for tumor suppression. Lastly, deficiencies in the origin licensing machinery will be discussed in relation to their influence on stem cell maintenance and human diseases.