Purpose: Triple-negative breast cancer (TNBC) is a refractory type of breast cancer with poor prognosis and limited choice for treatment. Previous studies had shown that TNBC has high expressions of transmembrane prostate androgen-induced protein (TMEPAI). TMEPAI was known to be induced by TGF-β/Smad signaling and have tumorigenic functions that converting TGF-β from tumor suppressor to tumor promoter and inducing epithelialmesenchymal transition (EMT). Therefore, we aimed to define the role of TMEPAI in triple-negative breast cancer cells treatment using several anti-cancers in the presence of TGF-β. Methods: TMEPAI-knock out (KO) was carried out in a triple-negative breast cancer cell, BT549. TMEPAI editing was developed using the CRISPR-Cas9 system using two combinations of sgRNA to remove exon 4 of the TMEPAI gene entirely. Genotyping and proteomic analysis were performed to check the establishment of the TMEPAI-KO cells. Wild type (WT) and KO cells were used to determine inhibitory concentration 50% (IC 50) of several anti-cancers: doxorubicin, cisplatin, paclitaxel, and bicalutamide in the presence of TGF-β treatment. Results: KO cells were successfully established by completely removing the TMEPAI gene, which was proven in genomic and proteomic analysis. Further, in TMEPAI-KO cells, we found a significant reduction of IC 50 for doxorubicin and paclitaxel, and minimal effects were seen for cisplatin and bicalutamide. Our findings suggest that TGF-β-induced TMEPAI attenuates the response of TNBC to doxorubicin and paclitaxel, but not to cisplatin and bicalutamide. Conclusion: TGF-β induced TMEPAI contributes to the reduced response of TNBC treatment to doxorubicin and paclitaxel, but minimal on cisplatin and bicalutamide. Further study is needed to confirm our findings in other growth factor-induced cells, as well as in in vivo model.
PMEPA1 (prostate transmembrane protein, androgen-induced 1)/TMEPAI (transmembrane prostate androgen-induced protein) is highly expressed in diverse cancers, including breast, lung and prostate cancers. It consists of four isoforms with distinct extracellular regions (isoforms a-d). The expression and function of these isoforms are still poorly understood. Hence, we aimed to identify the preferentially expressed isoforms in breast cancer cells and analyze possible differences in tumorigenic functions. In this study, we used 5′ Rapid Amplification of cDNA Ends (RACE) and Western blot analyses to identify the mRNA variants and protein isoforms of TMEPAI and found that TMEPAI isoform d as the major isoform expressed by TGF-β stimulation in breast cancer cells. We then generated CRISPR/ Cas9-mediated TMEPAI knockout (KO) breast cancer cell lines and used a lentiviral expression system to complement each isoform individually. Although there were no clear functional differences between isoforms, double PPxY (PY) motifs and a Smad-interaction motif (SIM) of TMEPAI were both essential for colony and sphere formation. Collectively, our results provide a novel insight into TMEPAI isoforms in breast cancer cells and showed that coordination between double PY motifs and a SIM of TMEPAI are essential for colony and sphere formation but not for monolayer cell proliferation. K E Y W O R D Sbreast cancer, isoform, PY motifs, SIM, TMEPAI |Genes to Cells PUTERI ET al.
Introduction Epithelial–mesenchymal transition (EMT) and overexpression of drug efflux transporters have been reported to cause doxorubicin resistance. Our previous study indicated that TMEPAI (transmembrane prostate androgen-induced protein) attenuated doxorubicin sensitivity in triple-negative breast cancer cells. However, how TMEPAI contributes to doxorubicin resistance in TNBC remains unclear. Thus, the present study aimed to elucidate the mechanism of TMEPAI in doxorubicin resistance in triple-negative breast cancer cells. Methods We used BT549, triple-negative cells wild type (WT), and BT549 TMEPAI knock-out. Both cells were treated with TGF-β 2 ng/mL for 24 hours, followed by TGF-β 2 ng/mL and doxorubicin 12.9 nM for another 24 hours. Afterward, the cells were harvested and counted. Cells were further lysed and used for RT-PCR and Western blot analysis. We determined the expression levels of proliferation, apoptosis, EMT markers, and drug efflux transporters. Additionally, we investigated the expressions of PI3K as well as SMAD3 and AKT phosphorylation. Results TNBC cells were shown to be less sensitive to doxorubicin in the presence of TMEPAI. TMEPAI was shown to alleviate the mRNA expressions of apoptosis markers: Bax, Bcl2, Caspase-3, and Caspase-9. Our results indicated that the presence of TMEPAI greatly amplifies EMT and increases drug efflux transporter expressions after doxorubicin treatment. Furthermore, our findings demonstrated that TMEPAI reduced the action of doxorubicin in inhibiting SMAD3 phosphorylation. TMEPAI was also shown to modify the effect of doxorubicin by reducing PI3K expressions and Akt phosphorylation in triple-negative breast cancer cells. Conclusion Our findings indicate that TMEPAI promotes EMT and drug efflux transporters at least in part by shifting doxorubicin action from SMAD3 phosphorylation reduction to PI3K/AKT inhibition in triple-negative breast cancer cells.
AIM: This study was aimed to determine the antifibrotic activity of Phaleria macrocarpa (PM) extract in liver fibrosis (LF) and its possible mechanism in the rat model. METHODS: Sprague Dawley male rats were injected with 2 mL/kg BW of carbon tetrachloride intraperitoneally twice a week for 2 weeks, followed by 1 mL/kg BW for 6 weeks. Afterward, the treatments began from the 3rd week: Silymarin 100 mg/kg BW/day, standardized PM extract (Proliverenol) 75 or 150 mg/kg BW/day orally. Rats were sacrificed in the 8th week. Blood and liver were collected to analyze liver function, liver damage and fibrosis marker, oxidative stress markers, pro-fibrogenic cytokine, and antifibrotic marker. RESULTS: Our study showed that the treatment of silymarin and PM resulted in the normalized activity of liver function, followed by the amelioration of oxidative stress, demonstrated by the decreased malondialdehyde levels and an increased ratio of glutathione and glutathione disulfide. All markers examined showed that PM extract has antioxidant activity due to decreased hepatic stellate cell activation. We also found a decrease in tumor growth factors-β1 and protein expressions of matrix metalloproteinases-13 in all treatment groups compared to the carbon tetrachloride group. There were tendencies of the decreased fibrotic area following improvements of biochemical parameters. CONCLUSION: PM extracts ameliorate carbon tetrachloride-induced LF. The proposed mechanism is by overcoming oxidative stress and regulating pro-fibrogenic cytokine and antifibrotic markers.
Cardiometabolic disorders (CMD) have become a global emergency and increasing burden on health and economic problems. Due to the increasing need for new drugs for cardiometabolic diseases, many alternative medicines from plants have been considered and studied. Moringa oleifera Lam. (MO), one of the native plants from several Asian countries, has been used empirically by people for various kinds of illnesses. In the present systematic review, we aimed to investigate the recent studies of MO in CMD and its possible mechanism of action. We systematically searched from three databases and summarized the data. This review includes a total of 108 papers in nonclinical studies and clinical trials of MO in cardiometabolic-related disorders. Moringa oleifera, extracts or isolated compound, exerts its effect on CMD through its antioxidative, anti-inflammatory actions resulting in the modulation in glucose and lipid metabolism and the preservation of target organ damage. Several studies supported the beneficial effect of MO in regulating the gut microbiome, which generates the diversity of gut microbiota and reduces the number of harmful bacteria in the caecum. Molecular actions that have been studied include the suppression of NF-kB translocation, upregulation of the Nrf2/Keap1 pathway, stimulation of total antioxidant capacity by reducing PKCζ activation, and inhibiting the Nox4 protein expression and several other proposed mechanisms. The present review found substantial evidence supporting the potential benefits of Moringa oleifera in cardiovascular or metabolic disorders.
Objective: This study aimed to investigate whether quercetin is able to improve the efficacy of sorafenib in triple negative breast cancer cells and explore the possibility of drug efflux transporters modulation by quercetin. Methods: We exposed MDA-MB-231, a triple negative breast cancer cell line, to several groups: sorafenib alone, quercetin alone, a combination of sorafenib-quercetin, and control. We determined cell viability over control weekly up to 4 w. At the end of the fourth week, mRNA expressions of drug efflux transporters (P-glycoprotein and breast cancer resistance protein [BCRP] and MRP2 [multidrug resistance-associated protein-2]) were examined. Results: Sorafenib alone was shown to maintain its efficacy for only two weeks, while quercetin alone was able to maintain its effect for four weeks. A combination of sorafenib-quercetin showed the best cytotoxicity effects compared with sorafenib or quercetin alone and was able to maintain its efficacy for four weeks. There were increased mRNA expressions of P-glycoprotein, BCRP, and MRP2 after four weeks of treatment with sorafenib, while treatment with quercetin decreased the drug efflux transporters expressions. A combination of sorafenib-quercetin decreased the mRNA expressions of both P-glycoprotein and BCRP, compared with sorafenib alone. Conclusion: We suggest that decreased expressions of both drug efflux transporters, P-glycoprotein and BCRP, mediated by quercetin ameliorate the efficacy of sorafenib in TNBC. Therefore, the addition of quercetin to sorafenib might be useful in the future in improving the therapeutic efficacy of sorafenib in triple negative breast cancer.
Objectives: In various liver disease models, including those for alcoholic liver diseases, curcumin, a polyphenolic compound derived fromCurcuma longa, is known to have an hepatoprotective effect. However, the mechanism of action underlying its effects on alcohol-induced hepaticfibrosis remains unknown. We aimed to investigate the mechanisms of action underlying the effects of curcumin, mainly involving the transforminggrowth factor (TGF)-β/Smad pathway.Methods: Hepatic stellate cells (HSCs), LX2, were incubated with 50 mM ethanol with or without curcumin (1 and 10 μM). Viable HSCs were countedusing a LUNATM automated cell counter, whereas the expressions of TGF-β, Smad3, tissue inhibitor of metalloproteinases-1 (TIMP-1), and type 1collagen mRNA were measured using quantitative reverse transcriptase polymerase chain reactions.Results: Curcumin significantly suppressed ethanol-induced HSCs proliferation. The antiproliferative effect of curcumin appeared to be dosedependent. In addition, the mRNA expressions of TGF-β, Smad3, TIMP-1, and type 1 collagen decreased in the cells treated with curcumin.Conclusion: Curcumin seems to attenuate ethanol-induced HSCs proliferation through the suppression of TGF-β and appears to reduce the productionof extracellular matrix as shown by the decreased expression of type 1 collagen.
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