Sulforaphane-cisplatin combination inhibits the stemness and metastatic potential of TNBCs via down regulation of sirtuins-mediated EMT signaling axis

BackgroundSulforaphane (SFN) is a naturally occurring organosulfur compound found in cruciferous vegetables such as broccoli, brussels sprouts and cabbage. SFN is known for its multiple therapeutic properties, such as HDAC inhibitory, chemo preventive and anti-cancer effects. Cisplatin (CIS) has limited effect against metastatic triple-negative breast cancer (TNBC). Additionally, CIS impose severe side effects to normal cells, and later TNBC cells develops resistance. Studies suggest that the overexpression of sirtuins (SIRTs) promotes CIS resistance and metastasis by activating epithelial-to-mesenchymal transition (EMT) pathway in TNBC.PurposeIn view of the above information, we investigated the therapeutic efficacy of SFN, in combination with CIS against TNBC metastasis and CIS resistance.MethodsThe anti-cancerous effect of SFN-CIS combination on human TNBC cell lines was demonstrated by utilizing MTT assay and, apoptosis and cell cycle assay followed by FACS analysis. The synergistic effect of SFN-CIS combination on the experimental metastasis was demonstrated by utilizing migration, invasion, chemotaxis, mammosphere and colony formation assay on human TNBC MDA-MB-231 and MDA-MB-468 cells. The role of SIRTs-mediated EMT signaling axis in the metastasis and chemoresistance was investigated by western blotting technique as well as sirtuin activity tests. This was further validated by using Chromatin immunoprecipitation (ChIP) analysis.ResultsWe found that SFN-CIS combination synergistically inhibits cellular growth of MDA-MB-231 and MDA-MB-468 cells. More importantly, SFN was found to protect normal kidney cells from CIS-induced toxicity. Further, SFN-CIS combination was found to synergistically inhibit metastatic-events via significantly altering EMT markers which was further associated with the suppression of SIRTs functions in TNBC cells. ChIP analysis validated that SFN-CIS combination suppresses EMT mechanism through altered chromatin modifications at E-cadherin promoter resulting in its re-expression.ConclusionThe results of the current study suggests that CIS when supplemented with SFN, inhibits metastasis and stemness potential of TNBC cells by down regulating SIRTs-mediated EMT cascade. Overall this study affirms that, this novel combination could be a promising strategy against SIRT-mediated TNBC metastasis and CIS-resistance.     

Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation,Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

Treatments for triple-negative breast cancer (TNBC) are limited; intermediate-conductance calcium-activated potassium (SK4) channels are closely involved in tumor progression, but little is known about these channels in TNBC. We aimed to investigate whether SK4 channels affect TNBC. First, by immunohistochemistry (IHC) and western blotting (WB), increased SK4 protein expression in breast tumor tissues was detected relative to that in non-tumor breast tissues, but there was no apparent expression difference between various subtypes of breast cancer (p>0.05). Next, functional SK4 channels were detected in the TNBC cell line MDA-MB-231 using WB, real-time PCR, immunofluorescence and patch-clamp recording. By employing SK4 specific siRNAs and blockers, including TRAM-34 and clotrimazole, in combination with an MTT assay, a colony-formation assay, flow cytometry and a cell motility assay, we found that the suppression of SK4 channels significantly inhibited cell proliferation and migration and promoted apoptosis in MDA-MB-231 cells (p<0.05). Further investigation revealed that treatment with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) caused MDA-MB-231 cells to undergo the epithelial-mesenchymal transition (EMT) and to show increased SK4 mRNA expression. In addition, the down-regulation of SK4 expression inhibited the EMT markers Vimentin and Snail1. Collectively, our findings suggest that SK4 channels are expressed in TNBC and are involved in the proliferation, apoptosis, migration and EMT processes of TNBC cells.     

PAK1 promotes proliferation,migration and invasion of hepatocellular carcinoma by facilitating EMT via directly up-regulating Snail

BackgroundHepatocellular carcinoma (HCC) is a primary cause of cancer mortality. PAK1 plays key roles in many types of cancers. However, the role of PAK1 in HCC is not clear.MethodsqRT-PCR and Western blotting were used to determine expressions of PAK1, Snail and epithelial mesenchymal transition (EMT)-related proteins. Luciferase reporter assay was used to measure the interaction between PAK1 and Snail. Wound healing, transwell, colony formation assays and flow cytometry were used to assess cell migration, invasion, proliferation and apoptosis. Mouse tumor xenograft model was used to determine the effect of PAK1 on tumor growth in vivo.ResultsPAK1 and Snail were up-regulated in HCC cells. PAK1 knockdown suppressed cell proliferation, migration and invasion, and increased apoptosis of HCC cells. PAK1 knockdown also inhibited tumor growth in vivo. Mechanistically, PAK1 promoted EMT by targeting Snail. Knockdown of PAK1 could up-regulate pro-apoptotic proteins but down-regulate proliferation-related proteins via suppressing β-catenin signaling pathway.ConclusionPAK1 promotes EMT process by increasing Snail, and facilitates progression of HCC by activating β-catenin pathway.     

LIPH promotes metastasis by enriching stem‐like cells in triple‐negative breast cancer

Lipase member H (LIPH), a novel member of the triglyceride lipase family. The clinical implications of its expression in breast cancer are still unclear. Therefore, in this study, we investigated the associations between LIPH and the tumorigenic behaviours of 144 triple‐negative breast cancer (TNBC) patients. The ratio and mammosphere‐forming ability of CD44+/CD24? stem‐like cells were tested. The role of LIPH in breast cancer cell migration and invasion was also evaluated. In addition, the effect of LIPH silencing on mitochondrial respiration was determined using the Seahorse assay. Finally, the effect of LIPH silencing on protein expression was determined via tandem mass tag‐based spectrometry and Western blotting. We found that LIPH expression was associated with metastasis in lymph nodes and distant organs (P = 0.025), resulting in poor survival among breast cancer patients (P = 0.027). LIPH knockdown significantly decreased both the ratio of CD44⁺/CD24^? stem‐like cells and their mammosphere‐forming ability. LIPH silencing promoted apoptosis, arrested cell cycle in the G2/M phase, mitigated the oxidation‐related oxygen consumption rate in the mitochondria, and reduced metabolism. LIPH inhibited adhesion between tumour cells and enhanced the epithelial‐mesenchymal transition. Tandem mass spectrometric analysis presented 68 proteins were differentially expressed in LIPH‐silenced cells and LIPH‐mediated modulation of tumour cell adhesion depended on integrin‐related CAPN2 and paxillin signalling. Overall, our findings provided strong evidence that LIPH up‐regulation promoted metastasis and the stemness of TNBC cells. Therefore, targeting LIPH is a potentially viable strategy for preventing metastasis in TNBC.     

MiR-210 in exosomes derived from CAFs promotes non-small cell lung cancer migration and invasion through PTEN/PI3K/AKT pathway

ObjectiveCancer-associated fibroblasts (CAFs) function as a crucial factor in tumor progression by carrying exosomes to neighboring cells. This study was assigned to expound the underlying mechanism of CAFs-derived exosomal miR-210 in non-small cell lung cancer (NSCLC) progression.MethodCAFs and normal fibroblasts (NFs) were isolated and identified. Exosomes secreted from CAFs and NFs were isolated to analyze their effects on tumor volume and epithelial-mesenchymal transition (EMT). Exosomal miR-210 expression level was measured. The effects of exosomal miR-210 and UPF1 on cell viability, EMT, PTEN/PI3K/AKT signal pathway were determined. Dual-luciferase reporter gene assay was utilized to validate the binding of UPF1 to miR-210.ResultsCAFs-derived exosomes (CAFs-exo) were successfully extracted and proven to be uptake by lung cancer cells. Up-regulated expression level of miR-210 was found in CAFs-exo, which was then proved to enhance cell migration, proliferation, invasion abilities and EMT in NSCLC cells. Overexpression of miR-210 can also inhibit UPF1 and PTEN, but activate the PTEN/PI3K/AKT pathway. UPF1 was a target gene of miR-210. MiR-210 can up-regulate UPF1 expression level to activate PTEN/PI3K/AKT pathway.ConclusionMiR-210 secreted by CAFs-exo could promote EMT by targeting UPF1 and activating PTEN/PI3K/AKT pathway, thereby promoting NSCLC migration and invasion.     

Atractylenolide I inhibits colorectal cancer cell proliferation by affecting metabolism and stemness via AKT/mTOR signaling

BackgroundAtractylenolide I (ATL-1) is a natural herbal compound used in traditional Chinese medicine that has exhibited anti-cancer properties. The anti-tumorigenic activity of ATL-1 against colorectal cancer (CRC) and the underlying signaling pathways involved in its mechanisms are examined here.HypothesisATL-1 exerts therapeutic effect against CRC by disrupting glucose metabolism and cancer stem cell maintenance via AKT/mTOR pathway regulation.Study designIn vitro studies were performed in COLO205 and HCT116 CRC cell lines and in vivo studies were conducted in a mouse xenograft model of CRC tumor.MethodsCRC cells were treated with ATL-1 at various concentrations, with or without inhibitors of AKT or mTOR. Cell proliferation, apoptosis, invasion, stemness maintenance, glucose metabolism, and AKT/mTOR signaling were evaluated. CRC tumor-xenografted mice were treated with an AKT inhibitor and/or ATL-1, and glucose metabolism and stemness maintenance were examined in tumor tissues.ResultsATL-1 significantly inhibited the invasion of CRC cells by inducing their apoptosis, possibly via the excessive production of reactive oxygen species. Glucose metabolism (Warburg effect) was also altered and stem-like traits were suppressed by ATL-1. In addition, ATL-1 effectively acted as an inhibitor or AKT/mTOR by downregulating the phosphorylation of proteins related to the AKT/mTOR pathway. In vivo studies showed that tumor weight and volume were reduced by ATL-1 and that aerobic glycolysis, stemness maintenance, and AKT/mTOR activation were impaired by ATL-1 in colorectal tumors.ConclusionsATL-1 acts as an effective agent to suppress colorectal tumor progression, mainly by inhibiting CRC cell proliferation through altering apoptosis, glucose metabolism, and stem-like behavior. These processes were mediated by the AKT/mTOR signaling pathway both in vitro and in vivo. ATL-1 may be a potential agent to be used in molecular-targeted strategies for cancer treatment.     

Combined Treatment with Exendin-4 and Metformin Attenuates Prostate Cancer Growth

Introduction

Recently, the pleiotropic benefits of incretin-based therapy have been reported. We have previously reported that Exendin–4, a glucagon-like peptide–1 (GLP–1) receptor agonist, attenuates prostate cancer growth. Metformin is known for its anti-cancer effect. Here, we examined the anti-cancer effect of Exendin–4 and metformin using a prostate cancer model.

Methods

Prostate cancer cells were treated with Exendin–4 and/or metformin. Cell proliferation was quantified by growth curves and 5-bromo–2′-deoxyuridine (BrdU) assay. TUNEL assay and AMP-activated protein kinase (AMPK) phosphorylation were examined in LNCaP cells. For in vivo experiments, LNCaP cells were transplanted subcutaneously into the flank region of athymic mice, which were then treated with Exendin–4 and/or metformin. TUNEL assay and immunohistochemistry were performed on tumors.

Results

Exendin–4 and metformin additively decreased the growth curve, but not the migration, of prostate cancer cells. The BrdU assay revealed that both Exendin–4 and metformin significantly decreased prostate cancer cell proliferation. Furthermore, metformin, but not Exendin–4, activated AMPK and induced apoptosis in LNCaP cells. The anti-proliferative effect of metformin was abolished by inhibition or knock down of AMPK. In vivo, Exendin–4 and metformin significantly decreased tumor size, and further significant tumor size reduction was observed after combined treatment. Immunohistochemistry on tumors revealed that the P504S and Ki67 expression decreased by Exendin–4 and/or metformin, and that metformin increased phospho-AMPK expression and the apoptotic cell number.

Conclusion

These data suggest that Exendin–4 and metformin attenuated prostate cancer growth by inhibiting proliferation, and that metformin inhibited proliferation by inducing apoptosis. Combined treatment with Exendin–4 and metformin attenuated prostate cancer growth more than separate treatments.     

基于PTEN-PI3K-AKT信号通路探讨竹节参总皂苷抑制人肺腺癌A549细胞增殖和转移的机制

目的:竹节参是人参属植物,和人参成分相似,前期研究其对肺癌具有一定的抑制作用,但作用机制不清,因此,本项目拟研究竹节参皂苷对人肺癌细胞系A549增殖、迁移和侵袭能力以及PTEN-PI3K-AKT信号通路的影响。方法:CCK8法测定不同浓度和不同作用时间的竹节参皂苷对A549存活率的影响,划痕实验测定细胞迁移能力,Transwell小室测定细胞的侵袭能力,ELISA试剂盒测定培养基上清中MMP-2和MMP-9水平的变化。Western blot测定PTEN、P-PI3K和P-Akt表达的变化。结果:竹节参皂苷对A549细胞增殖具有明显的抑制作用,呈浓度和时间依赖关系,与对照组比较具有统计学差异。同时,竹节参皂苷可以浓度依赖性的抑制细胞侵袭和转移,以及MMP-2和MMP-9细胞因子的分泌。Western blot结果表明竹节参皂苷可促进PTEN蛋白表达,抑制P-PI3K和P-Akt蛋白表达,采用PTEN的特异性抑制剂SF1670证实竹节参皂苷通过抑制PTEN发挥作用。结论:竹节参皂苷可抑制肺癌A549细胞增殖、迁移和侵袭,以及分泌蛋白MMP-2和MMP-9表达,其作用机制可能是通过调控PTEN抑制PI3K和Akt磷酸化,从而发挥抗癌作用。     

Shuganning injection,a traditional Chinese patent medicine,induces ferroptosis and suppresses tumor growth in triple-negative breast cancer cells

BackgroundTriple-negative breast cancer (TNBC), lacking targeted therapies currently, is susceptible to ferroptosis, a recently defined form of cell death.PurposeTo evaluate the anticancer activity of Shuganning injection (SGNI), a traditional Chinese patent medicine, on TNBC cells; To elucidate the mechanism of SGNI induced ferroptosis.MethodsThe anticancer activity of SGNI was examined via in vitro cell proliferation assays and in vivo xenograft growth assay. Ferroptosis was determined by flow-cytometric analysis of lipid ROS, labile iron pool measurement, and propidium iodide exclusion assay. The dependency on heme oxygenase 1 (HO-1) of SGNI induced ferroptosis was confirmed by genetic knockdown and pharmacological inhibition of the protein.ResultsSGNI selectively inhibited the proliferation of TNBC cells compared to non-TNBC breast cancer cells and normal cells. The cell death induced by SGNI in TNBC cells showed distinct morphology from apoptosis and could not be rescued by the pan-caspase inhibitor Z-VAD(OMe)-FMK. On the other hand, SGNI induced cell death was blocked by the lipid ROS scavengers ferrostatin-1 and liproxstatin-1, the acyl-CoA synthetase long chain family member 4 inhibitor rosiglitazone, and the iron chelators 1,10-phenanthroline and deferoxamine. These data indicated that SGNI induced a ferroptotic cell death of TNBC cells. Mechanistically, SGNI induced ferroptosis was dependent on HO-1, which promotes intracellular labile iron pool accumulation, and was alleviated by HO-1 knockdown and inhibition by tin protoporphyrin IX. In line with the in vitro data, SGNI significantly inhibited the xenograft growth of TNBC cell line MD-MB-231 in nude mice.ConclusionCollectively, our study elaborates on a promising regimen for TNBC treatment through induction of ferroptosis by SGNI, a traditional Chinese patent medicine currently available in the clinic, which merits further investigation.     

Simvastatin inhibits the development of radioresistant esophageal cancer cells by increasing the radiosensitivity and reversing EMT process via the PTEN-PI3K/AKT pathway

Acquired radioresistance compromises the efficacy of radiotherapy for carcinomas including esophageal cancer (EC), thus resulting in recurrence and poor survival. Recent research corroborated radiosensitive function of simvastatin in stem-like breast cancer cells. However, its role in EC radioresistance remains poorly elucidated. Here, we developed a radioresistant EC cell line Ec9706-R with higher resistance to irradiation relative to control Ec9706 cells. Intriguingly, Ec9706-R cells exhibited epithelial-mesenchymal transition (EMT) characteristics with high invasion and migration ability. Simvastatin sensitized radioresistance of Ec9706-R cells and suppressed cell proliferation, but aggravated radiation-induced apoptosis and caspase-3 activity. Furthermore, simvastatin reversed EMT and inhibited cell invasion and migration of Ec9706-R cells. Mechanism assay confirmed the activation of PI3K/AKT pathway after radiation, which was inhibited by simvastatin. After restoring this pathway by its activator, IGF-1, simvastatin-mediated radiosensitivity and EMT reversion were abrogated. Further assay substantiated the PTEN suppression after irradiation, which was elevated following simvastatin pre-treatment. Moreover, PTEN cessation attenuated the inhibitory effect of simvastatin on PI3K/AKT activation, and subsequently antagonized simvastatin-induced radiosensitivity and EMT reversion. Additionally, simvastatin aggravated radiation-mediated Ec9706-R tumor growth inhibition. Together, simvastatin inhibits the development of Ec9706-R cells by increasing radiosensitivity and reversing EMT via PTEN-PI3K/AKT pathway, implying a promising strategy against EC radioresistance.     

Melatonin inhibits cell growth and migration,but promotes apoptosis in gastric cancer cell line,SGC7901

Abstract

The pineal hormone, melatonin (MLT), has been shown to have therapeutic effects in patients with gastric cancer; however, the mechanisms for the anti-cancer effects are unknown. We investigated the effects of melatonin on cell proliferation, apoptosis, colony formation and cell migration in the gastric adenocarcinoma cell line, SGC7901, using MTT assay, Hoechst 33258 staining, flow cytometry, western blot, caspase-3 activity assay, soft agar colony formation assay, and scratch-wound assay. Our results showed that melatonin could inhibit cell proliferation, colony formation and migration efficiency, and it promoted apoptosis of SGC7901 cells. Our findings suggest that the anti-cancer effects of melatonin may be due to both inhibition of tumor cell proliferation and reduction of the metastatic potential of tumor cells.     

Glucose promotes breast cancer aggression and reduces metformin efficacy

Metformin treatment has been associated with a decrease in breast cancer risk and improved survival. Metformin induces complex cellular changes, resulting in decreased tumor cell proliferation, reduction of stem cells, and apoptosis. Using a carcinogen-induced rodent model of mammary tumorigenesis, we recently demonstrated that overfeeding in obese animals is associated with a 50% increase in tumor glucose uptake, increased proliferation, and tumor cell reprogramming to an “aggressive” metabolic state. Metformin significantly inhibited these pro-tumorigenic effects. We hypothesized that a dynamic relationship exists between chronic energy excess (glucose by dose) and metformin efficacy/action.

Media glucose concentrations above 5 mmol/L was associated with significant increase in breast cancer cell proliferation, clonogenicity, motility, upregulation/activation of pro-oncogenic signaling, and reduction in apoptosis. These effects were most significant in triple-negative breast cancer (TNBC) cell lines. High-glucose conditions (10 mmol/L or above) significantly abrogated the effects of metformin. Mechanisms of metformin action at normal vs. high glucose overlapped but were not identical; for example, metformin reduced IGF-1R expression in both the HER2+ SK-BR-3 and TNBC MDA-MB-468 cell lines more significantly at 5, as compared with 10 mmol/L glucose. Significant changes in gene profiles related to apoptosis, cellular processes, metabolic processes, and cell proliferation occurred with metformin treatment in cells grown at 5 mmol/L glucose, whereas under high-glucose conditions, metformin did not significantly increase apoptotic/cellular death genes. These data indicate that failure to maintain glucose homeostasis may promote a more aggressive breast cancer phenotype and alter metformin efficacy and mechanisms of action.     

Doxycycline inhibits the cancer stem cell phenotype and epithelial-to-mesenchymal transition in breast cancer

Experimental evidence suggest that breast tumors originate from breast cancer stem cells (BCSCs), and that mitochondrial biogenesis is essential for the anchorage-independent clonal expansion and survival of CSCs, thus rendering mitochondria a significant target for novel treatment approaches. One of the recognized side effects of the FDA-approved drug, doxycycline is the inhibition of mitochondrial biogenesis. Here we investigate the mechanism by which doxycycline exerts its inhibitory effects on the properties of breast cancer cells and BCSCs, such as mammosphere forming efficiency, invasion, migration, apoptosis, the expression of stem cell markers and epithelial-to-mesenchymal transition (EMT) related markers of breast cancer cells. In addition, we explored whether autophagy plays a role in the inhibitory effect of doxycycline on breast cancer cells. We find that doxycyline can inhibit the viability and proliferation of breast cancer cells and BCSCs, decrease mammosphere forming efficiency, migration and invasion, and EMT of breast cancer cells. Expression of stem cell factors Oct4, Sox2, Nanog and CD44 were also significantly downregulated after doxycycline treatment. Moreover, doxycycline could down-regulate the expression of the autophagy marker LC-3BI and LC-3BII, suggesting that inhibiting autophagy may be responsible in part for the observed effects on proliferation, EMT and stem cell markers. The potent inhibition of EMT and cancer stem-like characteristics in breast cancer cells by doxycycline treatment suggests that this drug can be repurposed as an anti-cancer drug in the treatment of breast cancer patients in the clinic.     

MicroRNA-10a promotes epithelial-to-mesenchymal transition and stemness maintenance of pancreatic cancer stem cells via upregulating the Hippo signaling pathway through WWC2 inhibition

MicroRNAs (miRNAs)-mediated cancer stem cells (CSCs) have drawn wide attention. This study aimed to probe the role of miR-10a in epithelial–mesenchymal transition (EMT) and stemness maintenance of pancreatic CSCs (PCSCs). Differentially expressed miRs and genes in pancreatic cancer (PC) were predicted via an online database, and the miR-10a and WW and C2 domain containing 2 (WWC2) expression were identified via a comparative study in PC and pancreatitis tissues. PCNCs were isolated and identified, and then the functional roles of miR-10a and WWC2 in proliferation, invasion, migration, self-renewal, colony formation abilities, EMT, and stemness maintenance of PCNCs were determined. The effects of miR-10a on tumor growth in vivo were studied by performing a xenograft tumor in nude mice. Consequently, miR-10a was highly expressed while WWC2 was lowly expressed in PC tissues. miR-10a could target WWC2 expression. miR-10a inhibition reduced EMT and stemness maintenance of PCSCs via enhancing WWC2 expression. The in vitro results were reproduced in in vivo studies. miR-10a promoted EMT and stemness maintenance of PCSCs via activating the Hippo signaling pathway. Our study provided evidence that miR-10a inhibition reduced EMT and stemness maintenance of PCSCs via upregulating WWC2 expression and inhibiting the Hippo signaling pathway.     

Sanguinarine impedes metastasis and causes inversion of epithelial to mesenchymal transition in breast cancer

BackgroundA large number of breast cancer patients perishes due to metastasis instead of primary tumor, but molecular mechanisms contributing towards cancer metastasis remain poorly understood. Therefore, prompting development of novel treatment is inevitable. A vast variety of plant derived natural substance possesses several therapeutically active constituents, e.g. alkaloids, flavonoids, tannins, resins, terpenoids etc. that exhibit various pharmacological properties e.g. anti-inflammatory, anti-microbial and anti-cancer properties. Sanguinarine (SAN) alkaloid found its place among such naturally occurring substances that exerts several pharmacological activities, including anti-cancer effects.PurposeUntil now, role of SAN not only against epithelial-mesenchymal transition (EMT) but also against metastasis progression in breast cancer remains indistinct. Thus, aim of the present study was to investigate effects of SAN on EMT process and cancer metastasis in animal model.MethodsMTT assay was performed to assess SAN effects on proliferation in breast cancer. Scratch assay was performed to evaluate effects of SAN on migration in breast cancer. Colony formation assay was performed to determine effects of SAN on colonization characteristics of breast cancer. Western blotting was performed to measure EMT regulating protein expression as well as major pathway protein expression induced against TGF-β treatment in breast cancer. Tail vein method of injecting breast cancer cells in bulb/c mice was conducted to study metastasis progression and thereafter assessing effects of SAN against metastasis in mice.ResultsIn vivo results: MTT assay performed, demonstrated dose dependent inhibition of cell proliferation in breast cancer. Scratch assay results showed, SAN played a major role as migration inhibitor in estrogen receptor positive (ER+) breast cancer. Colony forming assay results demonstrated that SAN constrains ability of breast cancer to develop into well-defined colonies. Western blotting results for EMT regulating protein expression, after TGF-β treatment showed, SAN inhibited cadherin switch in ER+ breast cancer. Moreover, expression of pathway proteins involved in EMT process after TGF-β treatment i.e. Smad, PI3K/Akt and MAP kinase were significantly masked against SAN treatment.In vivo resultsThe appearance of metastatic nodules in lung tissues of mice model, helps to study the effects of SAN against metastasis in bulb/c mice. The obtained results have confirmed that SAN impeded lung metastasis. The macroscopic examination has confirmed metastasis inhibitory role of SAN in breast cancer. The Hematoxylin and eosin (H&E) staining results further advocate anti-metastatic characteristics of SAN, presented by fewer metastatic nodule and lesions appearance in SAN treated mice compared to untreated metastasis mice.ConclusionIn summary, SAN displayed prominent anti-metastatic effects in animal model and anti-proliferation effects together with significant inhibitory potential on EMT regulating protein expression against TGF-β treatment in ER+ breast cancer. So, overall findings of our study highlighted the pre-clinical significance of SAN in animal model therefore, further studies in humans as a part of clinical trial will be needed to establish pharmacokinetics and other effects of SAN, so that it can be a potential candidate for future treatment of metastatic breast cancer (MBC).     

Role of transcription factor Sp1 in the 4-O-methylhonokiol-mediated apoptotic effect on oral squamous cancer cells and xenograft

Recently, biphenolic components derived from the Magnolia family have been studied for anti-cancer, anti-stress, and anti-inflammatory pharmacological effects. However, the pharmacological mechanism of action of 4-O-methylhonokiol (MH) is not clear in oral cancer. The aim of this study was to investigate the role of MH in apoptosis and its molecular mechanism in oral squamous cell carcinoma (OSCC) cell lines, HN22 and HSC4, as well as tumor xenografts. Here, we demonstrated that MH decreased cell growth and induced apoptosis in HN22 and HSC4 cells through the regulation of specificity protein 1 (Sp1). We employed several experimental techniques such as MTS assay, DAPI staining, PI staining, Annexin-V/7-ADD staining, RT-PCR, western blot analysis, immunocytochemistry, immunohistochemistry, TUNEL assay and in vivo xenograft model analysis. MH inhibited Sp1 protein expression and reduced Sp1 protein levels via both proteasome-dependent protein degradation and inhibition of protein synthesis in HN22 and HSC4 cells; MH did not alter Sp1 mRNA levels. We found that MH directly binds Sp1 by Sepharose 4B pull-down assay and molecular modeling. In addition, treatment with MH or knocking down Sp1 expression suppressed oral cancer cell colony formation. Moreover, MH treatment effectively inhibited tumor growth and Sp1 levels in BALB/c nude mice bearing HN22 cell xenografts. These results indicated that MH inhibited cell growth, colony formation and also induced apoptosis via Sp1 suppression in OSCC cells and xenograft tumors. Thus, MH is a potent anti-cancer drug candidate for oral cancer.