Resveratrol enhanced chemosensitivity by reversing macrophage polarization in breast cancer

BackgroundResveratrol, a naturally occurring polyphenolic compound, has been shown to inhibit cancer growth by targeting several cancer-related signalling pathways. In the tumor microenvironment (TME), tumor-associated macrophages (TAMs) are the most abundant leukocyte population that are associated with poor prognosis in over 80% of breast cancer cases. However, little is known about the effect of resveratrol in the TME.MethodsIn this study, MDA-MB-231(MB231), cisplatin resistance MDA-MB-231 (cisR), and T47D were used to examine the antitumor effect of resveratrol. The effectiveness of resveratrol, together with cisplatin as breast cancer treatment was investigated in vivo. Gene expressions of M1 (iNOS and CXCL10) and M2 (ARG1, CD163 and MRC1) markers in differentiated macrophages derived from THP-1 cells were examined to investigate the effect of resveratrol on TAM polarization in breast cancer progression.ResultsOur results demonstrated that resveratrol significantly reduced cell proliferation and enhanced chemosensitivity in breast cancer cells by inhibiting production of IL-6 and STAT3 activation. Treatment of resveratrol increased CXCL10 (M1 marker) expression. Further, resveratrol decreased IL-6 levels in LPS-treated differentiated macrophages. The use of resveratrol with cisplatin inhibited suppressed tumor growth when compared with cisplatin alone.ConclusionThis study revealed that resveratrol inhibited breast cancer cell proliferation by promoting M1/M2 macrophage polarization ratio and suppressing IL-6/pSTAT3 pathway.

Resveratrol enhanced chemosensitivity by reversing macrophage polarization in breast cancer.

BACKGROUND: Resveratrol, a naturally occurring polyphenolic compound, has been shown to inhibit cancer growth by targeting several cancer-related signalling pathways. In the tumor microenvironment (TME), tumor-associated macrophages (TAMs) are the most abundant leukocyte population that are associated with poor prognosis in over 80% of breast cancer cases. However, little is known about the effect of resveratrol in the TME. METHODS: In this study, MDA-MB-231(MB231), cisplatin resistance MDA-MB-231 (cisR), and T47D were used to examine the antitumor effect of resveratrol. The effectiveness of resveratrol, together with cisplatin as breast cancer treatment was investigated in vivo. Gene expressions of M1 (iNOS and CXCL10) and M2 (ARG1, CD163 and MRC1) markers in differentiated macrophages derived from THP-1 cells were examined to investigate the effect of resveratrol on TAM polarization in breast cancer progression. RESULTS: Our results demonstrated that resveratrol significantly reduced cell proliferation and enhanced chemosensitivity in breast cancer cells by inhibiting production of IL-6 and STAT3 activation. Treatment of resveratrol increased CXCL10 (M1 marker) expression. Further, resveratrol decreased IL-6 levels in LPS-treated differentiated macrophages. The use of resveratrol with cisplatin inhibited suppressed tumor growth when compared with cisplatin alone. CONCLUSION: This study revealed that resveratrol inhibited breast cancer cell proliferation by promoting M1/M2 macrophage polarization ratio and suppressing IL-6/pSTAT3 pathway.

Circulating cell-free miRNAs as biomarker for triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer in women globally. This subtype often has early and high recurrence rates resulting in poor survival, partially due to lack of targeted therapies. Therefore, there is an urgent need to identify TNBC-specific biomarkers for early diagnosis and treatment monitoring, and to develop more effective targeted therapy. METHODS: By using miRCURY LNA array platform, we compared the differential miRNA expressions in plasma of patient with TNBC (n=5) and non-TNBC (n=5), as well as healthy controls (n=5). Potential miRNAs were then validated in a large cohort of patients by real-time PCR. RESULTS: Ten putative miRNAs from the microarray data that differentially expressed between non-TNBC and healthy controls were identified. In the screening phase (n=90), we selected five miRNAs (miR-92a-3p, miR-342-3p, miR-16, miR-21 and miR-199a-5p) that could discriminate TNBC from non-TNBC for further validation. Results showed that miR-16, miR-21 and miR-199a-5p were underexpressed in TNBC when compared with non-TNBC, and were further validated in a large cohort (n=252). In addition, post-operative plasma levels of miR-16, miR-21 and miR-199a-5p were significantly restored when compared with pre-operative plasma of TNBC. Plasma miR-199a-5p expression in TNBC had significant difference when compared with non-TNBC and healthy controls, the receiver-operator characteristics curve analysis revealed the highest area under curve (AUC=0.8838) among all. The expression levels were associated with TNM stage and tumour subtypes. CONCLUSIONS: Our data suggest that miR-199a-5p could be a TNBC-specific marker with diagnostic value and provide insights into targeted therapy in the treatment of TNBC.

Warburg effect revisited: an epigenetic link between glycolysis and gastric carcinogenesis.

In cancer cells, glucose is often converted into lactic acid, which is known as the 'Warburg effect'. The reason that cancer cells have a higher rate of aerobic glycolysis, but not oxidative phosphorylation, remains largely unclear. Herein, we proposed an epigenetic mechanism of the Warburg effect. Fructose-1,6-bisphosphatase-1 (FBP1), which functions to antagonize glycolysis was downregulated through NF-kappaB pathway in Ras-transformed NIH3T3 cells. Restoration of FBP1 expression suppressed anchorage-independent growth, indicating the relevance of FBP1 downregulation in carcinogenesis. Indeed, FBP1 was downregulated in gastric carcinomas (P<0.01, n=22) and gastric cancer cell lines (57%, 4/7). Restoration of FBP1 expression reduced growth and glycolysis in gastric cancer cells. Moreover, FBP1 downregulation was reversed by pharmacological demethylation. Its promoter was hypermethylated in gastric cancer cell lines (57%, 4/7) and gastric carcinomas (33%, 33/101). Inhibition of NF-kappaB restored FBP1 expression, partially through demethylation of FBP1 promoter. Notably, Cox regression analysis revealed FBP1 promoter methylation as an independent prognosis predicator for gastric cancer (hazard ratio: 3.60, P=0.010). In summary, we found that NF-kappaB functions downstream of Ras to promote epigenetic downregulation of FBP1. Promoter methylation of FBP1 can be used as a new biomarker for prognosis prediction of gastric cancer. Such an important epigenetic link between glycolysis and carcinogenesis partly explains the Warburg effect.

Differential expression of microRNAs in plasma of patients with colorectal cancer: a potential marker for colorectal cancer screening.

OBJECTIVE: MicroRNAs (miRNAs) have been shown to offer great potential in the diagnosis of cancer. We investigated whether plasma miRNAs could discriminate between patients with and without colorectal cancer (CRC). METHODS: This study was divided into three phases: (1) marker discovery using real-time PCR-based miRNA profiling on plasma, corresponding cancerous and adjacent non-cancerous colonic tissues of five patients with CRC, along with plasma from five healthy individuals as controls; (2) marker selection and validation by real-time quantitative RT-PCR on a small set of plasma; and (3) independent validation on a large set of plasma from 90 patients with CRC, 20 patients with gastric cancer, 20 patients with inflammatory bowel disease (IBD) and 50 healthy controls. RESULTS: Of the panel of 95 miRNAs analysed, five were upregulated both in plasma and tissue samples. All the five miRNAs were validated on the plasma of 25 patients with CRC and 20 healthy controls. Both miR-17-3p and miR-92 were significantly elevated in the patients with CRC (p<0.0005). The plasma levels of these markers were significantly reduced after surgery in 10 patients with CRC (p<0.05). Further validation with an independent set of plasma samples (n = 180) indicated that miR-92 differentiates CRC from gastric cancer, IBD and normal subjects. This marker yielded a receiver operating characteristic curve area of 88.5%. At a cut-off of 240 (relative expression in comparison to RNU6B snRNA), the sensitivity was 89% and the specificity was 70% in discriminating CRC from control subjects. CONCLUSION: MiR-92 is significantly elevated in plasma of patients with CRC and can be a potential non-invasive molecular marker for CRC screening.

Dual inhibition of 5-LOX and COX-2 suppresses colon cancer formation promoted by cigarette smoke.

Previous studies indicate that the arachidonic acid-metabolizing enzymes COX-2 and 5-LOX are overexpressed during the process of colonic adenoma formation promoted by cigarette smoke. The aims of the present study were to investigate whether there exists a relationship between COX-2 and 5-LOX, and whether dual inhibition of COX-2 and 5-LOX has an anticarcinogenic effect in the colonic tumorigenesis promoted by cigarette smoke. Results showed that pretreating colon cancer cells with cigarette smoke extract (CSE) promoted colon cancer growth in the nude mouse xenograft model. Inhibition of COX-2 or 5-LOX reduced the tumor size. In the group treated with COX-2-inhibitor, the PGE2 level decreased while the LTB4 level increased. In contrast, in the 5-LOX-inhibitor treated group, the LTB4 level was reduced and the PGE2 level was unchanged. However, combined treatment with both COX-2 and 5-LOX inhibitors further inhibited the tumor growth promoted by CSE over treatment with either COX-2-inhibitor or 5-LOX-inhibitor alone. This was accompanied by the downregulation of PGE2 and LTB4. In an in vitro study, we found that the action of CSE on colon cancer cells was mediated by 5-LOX DNA demethylation. In summary, these results indicate that inhibition of COX-2 may lead to a shunt of arachidonic acid metabolism towards the leukotriene pathway during colonic tumorigenesis promoted by CSE. Suppression of 5-LOX did not induce such a shunt and produced a better response. Therefore, 5-LOX inhibitor is more effective than COX-2 inhibitor, and blocker of both COX-2 and 5-LOX may present a superior anticancer profile in cigarette smokers.

Effect of polysaccharides from Angelica sinensis on gastric ulcer healing.

Our previous study showed that a crude extract from Angelica sinensis (ASCE), which mainly consisted of polysaccharides, significantly promoted migration and proliferation of normal gastric epithelial cells. These results strongly suggest that ASCE has a direct wound healing effect on gastric mucosa. However, there is no report concerning the effect of ASCE on gastric ulcer healing in animal models. In this study, we found that ASCE promoted ulcer healing. The area of the ulcer was reduced. This was accompanied with a significant increase in mucus synthesis when compared with the control. Angiogenesis was inhibited by the treatment of ASCE. Cell proliferation, ODC and EGFR protein expression was not affected in this process. Thus, the mechanism of how ASCE accelerates ulcer healing in addition to its effect on mucus synthesis remains to be investigated.

A 3.0-kDa low molecular weight heparin promotes gastric ulcer healing in rats.

BACKGROUND: Previous studies have shown that intragastric administration of unfractionated heparin enhances gastric ulcer healing in rats. As the large molecule of heparin may be partially degraded in the upper gastrointestinal tract, it is likely that fragments of heparin, derived from the unfractionated parent compound, are involved in the anti-ulcer action in the stomach. Therefore, it is possible that low molecular weight heparin may have a similar ulcer healing effect. METHODS: Male Sprague-Dawley rats with acetic acid-induced gastric ulcers were given a 3.0-kDa low molecular weight heparin (0.6-6.0 mg/kg) intravenously or intragastrically once daily for 4 days. Ulcer healing, mucosal histological changes, angiogenesis and gastric mucus production both in vivo and in vitro were determined. The bleeding time was measured to indicate the anticoagulation activity. RESULTS: Both intravenous and intragastric low molecular weight heparin dose dependently accelerated gastric ulcer healing, which was accompanied by a significant increase in mucosal regeneration and proliferation, angiogenesis and mucus content in the stomach. The drug also stimulated the mucus production in MKN-28 cells. Drug administration by either route did not alter the bleeding time in rats. CONCLUSIONS: A 3.0-kDa low molecular weight heparin possesses an ulcer healing effect similar to that of unfractionated heparin in the stomach of the rat. This smaller molecular drug is superior to the unfractionated form, does not affect the coagulation activity and may show better absorption in the gastrointestinal tract.

Epidermal growth factor and its receptor in chronic active gastritis and gastroduodenal ulcer before and after Helicobacter pylori eradication.

BACKGROUND: Helicobacteria pylori infection of gastroduodenal mucosa is strongly associated with gastritis and peptic ulcer disease. The aims of the present study were to compare the gastroduodenal mucosal levels of epidermal growth factor (EGF) and its receptor (EGFR) among H. pylori-negative controls and H. pylori infected patients with chronic active gastritis or gastroduodenal ulcer before and after H. pylori eradication. METHODS: The protein levels of EGF in mucosal tissues and saliva were determined by a solid-phase enzyme-linked immunosorbent assay (ELISA). Repeat transcription-polymerase chain reaction and the following polymerase chain reaction ELISA were employed to examine the mucosal EGFR mRNA expression. RESULTS: Mucosal injury and H. pylori infection increased EGF protein levels and EGFR mRNA expression in the antral mucosa. The concentration of EGF in saliva was not affected by mucosal damage or H. pylori infection. Successful H. pylori eradication normalized the EGFR mRNA back to its basal level 6 weeks after treatment. However, after unsuccessful eradication their high levels in the antrum persisted. All patients experienced ulcer healing after drug treatment, regardless of H. pylori eradication. CONCLUSIONS: Mucosal damage increased the expression of EGF protein and EGFR mRNA in the gastric mucosa. H. pylori could induce the expression of EGFR but not the EGF in the antral mucosa. The expression of EGFR could be a contributing factor for ulcer healing in patients with H. pylori infection.

A mechanistic study of proliferation induced by Angelica sinensis in a normal gastric epithelial cell line.

It has been reported that an extract from Angelica sinensis mainly consisting of polysaccharides (95%) prevented ethanol- or indomethacin-induced gastric mucosal damage (Cho CH et al. Planta Med 2000;66:348-51). However, it is not known whether Angelica sinensis has a direct stimulatory effect on the healing of gastric mucosal lesions. To study the hypothesis that Angelica sinensis has a direct mucosal healing effect in rats and in isolated gastric epithelial cells, we assessed the wound repair in both animals and normal cell culture (RGM-1), as well as [3H]thymidine incorporation, ornithine decarboxylase (ODC) activity, and ODC protein and c-Myc protein expression after different treatments in RGM-1 cells. We found that Angelica sinensis crude extract (ASCE) dose-dependently enhanced gastric ulcer healing in rats and promoted wound repair in RGM-1 cells. It also significantly stimulated [3H]thymidine incorporation and ODC activity in RGM-1 cells in a concentration-dependent manner. ODC and c-Myc protein expression was also increased as a result of this process. DL-alpha-difluoromethyl-ornithine repressed the [3H]thymidine incorporation and ODC activity induced by ASCE. Pretreatment with c-Myc antisense oligodeoxynucleotides blocked the stimulatory action of ASCE on [3H]thymidine incorporation and ODC protein expression. These data suggest that ASCE has a direct mucosal healing effect on gastric epithelial cells, while ODC and c-Myc are closely associated with this effect.

Modulation of heme oxygenase in tissue injury and its implication in protection against gastrointestinal diseases.

Heme oxygenase (HO) is the rate-limiting enzyme in the catabolism of heme, followed by production of biliverdin, free iron and carbon monoxide (CO). There are three isoforms of HO: HO-1 is highly inducible, whereas HO-2 and HO-3 are constitutively expressed. In addition to heme, a variety of nonheme compounds, including heavy metals, cytokines, endotoxins and heat shock stress are strong inducers of HO-1 expression. Many studies indicated that induction of HO-1 is associated with a protective response due to the removal of free heme, which is shown to be toxic. However, recent studies demonstrated that the expression of HO-1 in response to different inflammatory mediators could contribute in part to the resolution of inflammation and have protective effects on brain, liver, kidney and lung against injuries. These beneficial effects seem to be due to the production of bile pigment biliverdin and bilirubin that is a potent antioxidant, as well as the release of iron and CO. However, there are few studies concerning the relationship between HO-1 and inflammation as well as injury in the gut. Interestingly, a preliminary study implicated that induction of HO-1 expression in a colonic damage model induced by trinitrobenzene sulfonic acid played a critical protective role, indicating that activation of HO-1 could act as a natural defensive mechanism to alleviate inflammation and tissue injury in the gastrointestinal tract.

Subcellular distribution of ankyrin in developing rabbit heart--relationship to the Na+-Ca2+ exchanger.

Ankyrins are a multigene family of proteins that function as adapters between the cytoskeleton and trans-membrane proteins, such as ion channels. Previous studies have shown the linkage between ankyrin and ionic transport proteins such as Na+-K+ ATPase, voltage-dependent Na+ channels and Ca2+ channels. In the present study, we have investigated the subcellular distribution of ankyrin and its relationship to the Na+-Ca2+ exchange protein in immature and adult rabbit ventricular myocytes. Isolated single cardiomyocytes from neonatal, juvenile and adult rabbit hearts were examined by immunofluorescence labeling techniques, using antibodies against ankyrin and the Na+-Ca2+ exchanger. We found that in neonatal rabbit cardiac myocytes, ankyrin labeling was mainly present at the Z disk, whereas the Na+-Ca2+ exchanger was only present on the peripheral sarcolemma. At 2 weeks of age, ankyrin labeling was still predominantly observed at the level of the Z disks as well as in the partially developed T-tubules. In the adult cells, however, ankyrin and the Na+-Ca2+ exchanger seem to be co-localized within T-tubules and at the costamere region of the peripheral sarcolemma. Immunogold labeling studies at the higher resolution electron microscopic level using cyrosection tissues of rabbit heart at different ages confirm these findings. These results indicate that the distribution pattern of ankyrin and the Na+-Ca2+ exchanger changes with development in rabbit ventricular myocytes.