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.     

IKKβ regulates the repair of DNA double-strand breaks induced by ionizing radiation in MCF-7 breast cancer cells

Activation of the IKK-NFκB pathway increases the resistance of cancer cells to ionizing radiation (IR). This effect has been largely attributed to the induction of anti-apoptotic proteins by NFκB. Since efficient repair of DNA double strand breaks (DSBs) is required for the clonogenic survival of irradiated cells, we investigated if activation of the IKK-NFκB pathway also regulates DSB repair to promote cell survival after IR. We found that inhibition of the IKK-NFκB pathway with a specific IKKβ inhibitor significantly reduced the repair of IR-induced DSBs in MCF-7 cells. The repair of DSBs was also significantly inhibited by silencing IKKβ expression with IKKβ shRNA. However, down-regulation of IKKα expression with IKKα shRNA had no significant effect on the repair of IR-induced DSBs. Similar findings were also observed in IKKα and/or IKKβ knockout mouse embryonic fibroblasts (MEFs). More importantly, inhibition of IKKβ with an inhibitor or down-regulation of IKKβ with IKKβ shRNA sensitized MCF-7 cells to IR-induced clonogenic cell death. DSB repair function and resistance to IR were completely restored by IKKβ reconstitution in IKKβ-knockdown MCF-7 cells. These findings demonstrate that IKKβ can regulate the repair of DSBs, a previously undescribed and important IKKβ kinase function; and inhibition of DSB repair may contribute to cance cell radiosensitization induced by IKKβ inhibition. As such, specific inhibition of IKKβ may represents a more effective approach to sensitize cancer cells to radiotherapy.     

Can vitamin A modify the activity of docetaxel in MCF-7 breast cancer cells?

Docetaxel is one of the most effective chemotherapeutic agents in the treatment of breast cancer. On the other hand, the vitamin A family compounds play the essential roles in many biological processes in mammary gland. The aim of our study was to investigate the effect of all-trans retinol, carotenoids (beta-carotene, lycopene) and retinoids (9-cis, 13-cis and all-trans retinoic acid) on the activity of docetaxel and to compare these effects with the estradiol and tamoxifen actions on human ER(+) MCF-7 breast cancer cell line. The evaluation was based on [3H] thymidine incorporation and the proliferative activity of PCNA and Ki 67 positive cells. In our study, the incorporation of [3H] thymidine into cancer cells was inhibited to 50% by 0.2, 0.5 and 1 microM of docetaxel in the 24-hour culture and addition of estradiol (0.001 microM) didn't influence the results. However, addition of tamoxifen caused a statistically significant decrease of the percentage of the proliferating cells in the culture medium with 0.2 and 0.5 microM of docetaxel (38.99 +/- 2.84%, p<0.01 and 40.67 +/- 5.62%, p<0.01) in comparison to the docetaxel only group. The above-mentioned observations were also confirmed with the use of the immunohistochemical investigations. Among the examined vitamin A family compounds, the simultaneous application of beta-carotene (0.1 microM) and docetaxel (0.2 microM) resulted in a statistically significant reduction in the percentage of proliferating cells (40.25 +/- 14.62%, p<0.01). Lycopene (0.1 microM), which stimulates the growth of breast cancer cells in a 24-hour culture, had an inhibitory effect (42.97 +/- 9.58%, p<0.01) when combined with docetaxel (0.2 microM). Although, beta-carotene and lycopene belong to the different chemical groups, they surprisingly had a similar inhibitory influence on both growth and proliferation of MCF-7 breast cancer cells when combined with docetaxel. The application of docetaxel either with beta-carotene or lycopene had comparable inhibitory effect on breast cells growth and proliferation as tamoxifen. Therefore, it may suggest a possible important role of these carotenoids in the breast cancer therapy in women especially when docetaxel is applied.     

MiR-125b regulates the proliferation and metastasis of triple negative breast cancer cells via the Wnt/β-catenin pathway and EMT

Background/aim: MiR-125b plays an important role in breast cancer. The current study was to explore the expression and function of miR-125b in triple negative breast cancer cells. Materials and methods: The expression of miR-125b in human TNBC samples and cell lines were examined by qRT-PCR. MTT, scratch assays and transwell assays were utilized to observe the proliferation, migration and invasion ability. MiR-125b’s target gene and downstream signaling pathways were investigated by Luciferase Reporter Assays, qRT-PCR, immunofluorescence assays and western bolt. Results: MiR-125b was highly expressed in human TNBC tissues and cell lines. Inhibiting miR-125b expression suppressed the proliferation, cell migration and invasion. The three-prime untranslated region (3´-UTR) of adenomatous polyposis coli (APC) mRNA contains miR-125b binding sites, and inhibiting miR-125b expression suppressed the activity of the intracellular Wnt/β-catenin pathways and EMT. Conclusion: Inhibiting miR-125b regulates the Wnt/β-catenin pathway and EMT to suppress the proliferation and migration of MDA-MB-468 TNBC cells.     

β-Lactam type molecular scaffolds for antiproliferative activity: Synthesis and cytotoxic effects in breast cancer cells

A series of novel β-lactam containing compounds are described as antiproliferative agents and potential selective modulators of the oestrogen receptor. The purpose of the study is to evaluate the antiproliferative effects of these compounds on human MCF-7 and MDA MB-231 breast cancer cells. The compounds are designed to contain three aryl ring substituents arranged on the heterocyclic azetidin-2-one (β-lactam), thus providing conformationally restrained analogues of the triarylethylene arrangement exemplified in the tamoxifen type structure. The compounds demonstrated potency in antiproliferative assays against MCF-7 human breast cancer cell line at low micromolar to nanomolar concentrations with low cytotoxicity and moderate binding affinity to the oestrogen receptor. The effect of a number of aryl and amine functional group substitutions on the antiproliferative activity of the β-lactam products was explored and a brief computational structure–activity relationship investigation with molecular simulation was investigated.     

Can transforming growth factor-beta1 and retinoids modify the activity of estradiol and antiestrogens in MCF-7 breast cancer cells?

Retinoic acid and transforming growth factor-beta (TGF-beta) affect differentiation, proliferation and carcinogenesis of epithelial cells. The effect of both compounds on the proliferation of cells of the hormone sensitive human breast cancer cell line (ER+) MCF-7 was assessed in the presence of estradiol and tamoxifen. The assay was based on [3H]thymidine incorporation and the proliferative activity of PCNA- and Ki 67-positive cells. The apoptotic index and expression of the Bcl-2 and p53 antigens in MCF-7 cells were also determined. Exogenous TGF-beta1 added to the cell culture showed antiproliferative activity within the concentration range of 0.003-30 ng/ml. Irrespective of TGF-beta1 concentrations, a marked reduction in the stimulatory action of estradiol (10(-9) and 10(-8) M) was observed whereas in combination with tamoxifen (10(-7) and 10(-6) M) only 30 ng/ml TGF-beta1 caused a statistically significant reduction to approximately 30% of the proliferative cells. In further experiments we examined the effect of exposure of breast cancer cells to retinoids in combination with TGF-beta1. The incorporation of [3H]thymidine into MCF-7 cells was inhibited to 52 +/- 19% (control =100%) by 3 ng/ml TGF-beta1, and this dose was used throughout. It was found that addition of TGF-beta1 and isotretinoin to the culture did not decrease proliferation, while TGF-beta1 and tretinoin at low concentrations (3 x 10(-8) and 3 x 10(-7) M) reduced the percentage of proliferating cells by approximately 30% (67+/-8% and 67+/-5%, P<0.05 compared to values in the tretinoin group). Both retinoids also led to a statistically significant decrease in the stimulatory effect of 10(-9) M estradiol, attenuated by TGF-beta1. In addition, the retinoids in combination with TGF-beta1 and tamoxifen (10(-6) M) caused a further reduction in the percentage of proliferating cells. Immunocytochemical analysis showed that all the examined compounds gave a statistically significant reduction in the percentage of cells with a positive reaction to PCNA and Ki 67 antigen. TGF-beta1, isotretinoin and tretinoin added to the culture resulted in the lowest percentage of PCNA positive cells. However, the lowest fraction of Ki 67 positive cells was observed after addition of isotretinoin. The obtained results also confirm the fact that the well-known regulatory proteins Bcl-2 and p53 play an important role in the regulation of apoptosis in the MCF-7 cell line, with lowered Bcl-2 expression accompanying easier apoptotic induction. The majority of the examined compounds act via the p53 pathway although some bypass this important proapoptotic factor.     

Inhibition of integrin β3, a binding partner of kallistatin,leads to reduced viability,invasion and proliferation in NCI-H446 cells

Background

Kallistatin is a serine proteinase inhibitor and heparin-binding protein. It is considered an endogenous angiogenic inhibitor. In addition, multiple studies demonstrated that kallistatin directly inhibits cancer cell growth. However, the molecular mechanisms underlying these effects remain unclear.

Methods

Pull-down, immunoprecipitation, and immunoblotting were used for binding experiments. To elucidate the mechanisms, integrin β3 knockdown (siRNA) or blockage (antibody treatment) on the cell surface of small the cell lung cancer NCI-H446 cell line was used.

Results

Interestingly, kallistatin was capable of binding integrin β3 on the cell surface of NCI-H446 cells. Meanwhile, integrin β3 knockdown or blockage resulted in loss of antitumor activities induced by kallistatin. Furthermore, kallistatin suppressed tyrosine phosphorylation of integrin β3 and its downstream signaling pathways, including FAK/-Src, AKT and Erk/MAPK. Viability, proliferation and migration of NCI-H446 cells were inhibited by kallistatin, with Bcl-2 and Grb2 downregulation, and Bax, cleaved caspase-9 and caspase 3 upregulation.

Conclusions

These findings reveal a novel role for kallistatin in preventing small cell lung cancer growth and mobility, by direct interaction with integrin β3, leading to blockade of the related signaling pathway.

     

Furanodiene enhances tamoxifen-induced growth inhibitory activity of ERa-positive breast cancer cells in a PPARγ independent manner

Herbal plants are enriched with compounds with a wide range of biological activities. Furanodiene is a sesquiterpene isolated from Rhizoma Curcumae. Growing evidence shows furanodiene exhibits diversified activities of hepatoprotection, anti-inflammation, anti-angiogenesis, and anti-tumor. However, its biological activities against breast cancer have not been deeply understood, and its potential as an anti-breast cancer agent combined with tamoxifen (TAM) has not been evaluated so far. This study describes the combined effects of furanodiene and TAM in human breast cancer cells in vitro. The results showed that ERa-negative MDA-MB-231 cells were much more sensitive than ERa-positive MCF-7 cells to the growth inhibition due to furanodiene. Combined administration of furanodiene and TAM led to marked increase in growth inhibition, cell cycle arrest and pro-apoptotic activity in ERa-positive cells compared to individual agent, and enhanced the down-regulation of p-cyclin D1, cyclin D1, CDK2, CDK6, p-Rb, Rb and p-p44, and the up-regulation of p27, Bax and Bad, but did not show increased cytotoxicity in ERa-negative MCF-10A non-tumorigenic breast epithelial cells. Co-incubation induced the typical PARP cleavage or caspase 9 cleavages compared to individual agent. In addition, PPARγ activity inhibition by its antagonist T0070907 did not significantly reverse the enhanced effect of furanodiene and TAM suggesting that anti-cancer properties of combination were PPARγ independent. Our data indicated that furanodiene could enhance the growth inhibitory and pro-apoptotic activity of TAM by inducing cell cycle arrest and cell apoptosis via CDKs-cyclins and mitochondria-caspases-dependent, and PPARγ-independent signaling pathways in breast cancer cells, without contributions to the cytotoxicity of TAM.     

Wnt/β-catenin signaling regulates cancer stem cells in lung cancer A549 cells

Wnt/β-catenin signaling plays an important role not only in cancer, but also in cancer stem cells. In this study, we found that β-catenin and OCT-4 was highly expressed in cisplatin (DDP) selected A549 cells. Stimulating A549 cells with lithium chloride (LiCl) resulted in accumulation of β-catenin and up-regulation of a typical Wnt target gene cyclin D1. This stimulation also significantly enhanced proliferation, clone formation, migration and drug resistance abilities in A549 cells. Moreover, the up-regulation of OCT-4, a stem cell marker, was observed through real-time PCR and Western blotting. In a reverse approach, we inhibited Wnt signaling by knocking down the expression of β-catenin using RNA interference technology. This inhibition resulted in down-regulation of the Wnt target gene cyclin D1 as well as the proliferation, clone formation, migration and drug resistance abilities. Meanwhile, the expression of OCT-4 was reduced after the inhibition of Wnt/β-catenin signaling. Taken together, our study provides strong evidence that canonical Wnt signaling plays an important role in lung cancer stem cell properties, and it also regulates OCT-4, a lung cancer stem cell marker.