Breast cancer is a collection of diseases in which molecular phenotypes can act as both indicators and mediators of therapeutic strategy. Therefore, candidate therapeutics must be assessed in the ...context of multiple cell lines with known molecular phenotypes. Docosahexaenoic acid (DHA) and curcumin (CCM) are dietary compounds known to antagonize breast cancer cell proliferation. We report that these compounds in combination exert a variable antiproliferative effect across multiple breast cell lines, which is synergistic in SK-BR-3 cells and triggers cell signaling events not predicted by the activity of either compound alone.
Dose response curves for CCM and DHA were generated for five breast cell lines. Effects of the DHA+ CCM combination on cell proliferation were evaluated using varying concentrations, at a fixed ratio, of CCM and DHA based on their individual ED₅₀. Detection of synergy was performed using nonlinear regression of a sigmoid dose response model and Combination Index approaches. Cell molecular network responses were investigated through whole genome microarray analysis of transcript level changes. Gene expression results were validated by RT-PCR, and western blot analysis was performed for potential signaling mediators. Cellular curcumin uptake, with and without DHA, was analyzed via flow cytometry and HPLC.
CCM+DHA had an antiproliferative effect in SK-BR-3, MDA-MB-231, MDA-MB-361, MCF7 and MCF10AT cells. The effect was synergistic for SK-BR-3 (ER⁻ PR⁻ Her2⁺) relative to the two compounds individually. A whole genome microarray approach was used to investigate changes in gene expression for the synergistic effects of CCM+DHA in SK-BR-3 cells lines. CCM+DHA triggered transcript-level responses, in disease-relevant functional categories, that were largely non-overlapping with changes caused by CCM or DHA individually. Genes involved in cell cycle arrest, apoptosis, inhibition of metastasis, and cell adhesion were upregulated, whereas genes involved in cancer development and progression, metastasis, and cell cycle progression were downregulated. Cellular pools of PPARγ and phospho-p53 were increased by CCM+DHA relative to either compound alone. DHA enhanced cellular uptake of CCM in SK-BR-3 cells without significantly enhancing CCM uptake in other cell lines.
The combination of DHA and CCM is potentially a dietary supplemental treatment for some breast cancers, likely dependent upon molecular phenotype. DHA enhancement of cellular curcumin uptake is one potential mechanism for observed synergy in SK-BR-3 cells; however, transcriptomic data show that the antiproliferation synergy accompanies many signaling events unique to the combined presence of the two compounds.
Metastasis is the leading cause of death from breast cancer. A major factor of metastasis is the migration of cancerous cells to other tissues by way of up-regulated chemokine receptors, such as ...CXCR4, on the cell surface. Much is known of the beneficial effects of omega-3 polyunsaturated fatty acids (n-3 PUFA) on cancer; however, the mechanisms behind these effects are unclear. For this study, we investigated the effects of two n-3 PUFAs, docosahexaenoic acid and eicosapentaenoic acid, on CXCR4 expression and activity in the MDA-MB-231 breast cancer cell line. We compared the n-3 PUFAs with the saturated fatty acid stearic acid as a control. Treatment of the cells with n-3 PUFAs resulted in reduced surface expression of CXCR4, but had no effect on overall CXCR4 expression. Consequently, we found that the fatty acid treatment significantly reduced CXCR4-mediated cell migration. Successful CXCR4-mediated signaling and migration requires the cholesterol-rich membrane microdomains known as lipid rafts. Treatment with n-3 PUFAs disrupted the lipid raft domains in a manner similar to methyl-beta-cyclodextrin and resulted in a partial displacement of CXCR4, suggesting a possible mechanism behind the reduced CXCR4 activity. These results were not observed in cells treated with stearic acid. Together, our data suggest that n-3 PUFAs may have a preventative effect on breast cancer metastasis in vitro. This suggests a previously unreported potential benefit of n-3 PUFAs to patients with metastatic breast cancer. The data presented in this study may also translate to other disorders that involve up-regulated chemokine receptors.
Epidemiological studies have linked fish oil consumption to a decreased incidence of cancer. The anticancer effects of fish oil are mostly attributed to its content of omega‐3 fatty acids: ...eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). However, DHA, because of its unique effect of altering membrane composition, is often regarded as the major omega‐3 fatty acid involved in anticancer activity. Although use of DHA as an anticancer drug to prevent or treat human cancer in clinical settings has not yet been well established, recent studies suggest that DHA can be very effective as an adjuvant with other anticancer agents. In this article, we present studies that show the role of DHA in improving anticancer drug efficacy. Several in vitro and animal studies suggest that combining DHA with other anticancer agents often improves efficacy of anticancer drugs and also reduces therapy‐associated side effects. Incorporation of DHA in cellular membranes improves drug uptake, whereas increased lipid peroxidation is another mechanism for DHA‐mediated enhanced efficacy of anticancer drugs. In addition, several intracellular targets including cyclooxygenase‐2, nuclear factor kappa B, peroxisome proliferator‐activated receptor gamma, mitogen‐activated protein kinase, AKT, and BCL‐2/BAX are found to play an important role in DHA‐mediated additive or synergistic interaction with anticancer drugs. The data suggest that DHA is a safe, natural compound that can greatly improve the anticancer properties of anticancer drugs. Use of DHA with anticancer treatments provides an avenue to therapeutic improvement that involves less risk or side effects for patients and reduced regulatory burden for implementation.
Multiple myeloma (MM) remains an incurable malignancy indicating a need for continued investigation of novel therapies. Recent studies have highlighted the role of cyclin-dependent kinases (CDK) in ...the pathogenesis of MM. PD0332991 (Palbociclib) is an orally bioavailable, highly selective inhibitor of the CDK4/6-cyclin complex and downstream retinoblastoma protein (Rb) activation pathway that induces cell cycle arrest in the G1 phase.
In this review, the authors summarize the role of the CDK4/6 signaling pathway in MM. They also summarize the development of PD0332991 as a specific inhibitor of CDK4/6, and the reported preclinical and clinical data supporting the potential role of PD0332991 in MM.
While PD0332991 is essentially cytostatic, inducing prolonged G1 arrest, it enhances the cytotoxic effect of other agents effective in MM, including bortezomib and lenalidomide, as confirmed in early phase clinical trials. However, with a plethora of other drugs of different classes being tested in MM, further development of PD0332991 will depend on defining the most efficacious combination with least toxicity. An unexplored opportunity remains the potential protective effect of PD0332991 against lytic bone lesions of MM. The next few years are likely to better define the place of PD0332991 in the treatment of MM.
Abstract Molluscum contagiosum poxvirus (MCV) type 1 and type 2 encode two chemokine-like proteins MC148R1 and MC148R2. It is believed that MC148R proteins function by blocking the inflammatory ...response. However, the mechanism of the proposed biological activities of MC148R proteins and the role of the additional C-terminal cysteines that do not exist in other chemokines are not understood. Here, we demonstrated in two different assay systems that His-tagged MC148R1 displaces the interaction between CXCL12α and CXCR4. The N-terminal cysteines but not the additional C-terminal cysteines modulate this displacement. His-tagged MC148R1 blocked both CXCL12α-mediated and MIP-1α-mediated chemotaxis. In contrast, MC148R2 blocked MIP-1α-mediated but not CXCL12α-mediated chemotaxis. Immunoprecipitation by antibodies to MC148R1 or CXCL12α followed by immunoblotting and detection by antibodies to the other protein demonstrated physical interaction of His-tagged CXCL12α and His-tagged MC148R1. Interaction with chemokines might mask the receptor interaction site resulting in decreased binding and impairment of the biological activities.
► 2,6-Diisopropylphenyl–docosahexaenoamide conjugates (DIP–DHA) inhibits the proliferation of T-cell leukemic cell lines. ► DIP–DHA resulted in increased activation of caspase-3, and caspase-7. ► ...DIP–DHA significantly downregulated CXCR4 surface expression.
We have previously characterized the effects of 2,6-diisopropylphenyl–docosahexaenoamide (DIP–DHA) conjugates and their analogs on the proliferation and progression of breast cancer cell lines. For this study, we investigated the effects of the DIP–DHA conjugate on 2 representative T cell acute lymphoblastic leukemia (T-ALL) cell lines: CEM and Jurkat. Treatment of both cell lines with DIP–DHA resulted in significantly greater inhibition of proliferation and induction of apoptosis than that of parent compounds, 2,6-diisopropylphenol (DIP) or docosahexaenoate (DHA). Treatment of the cells with DIP–DHA resulted in increased activation of caspase-3, and caspase-7. Furthermore, induction of apoptosis in both cell lines was reversed in the presence of a caspase family inhibitor. Treatment with DIP–DHA reduced mitochondrial membrane potential. These observations suggest that the effects are driven by intrinsic apoptotic pathways. DIP–DHA treatment also downregulated surface CXCR4 expression, an important chemokine receptor involved in cancer metastasis that is highly expressed in both CEM and Jurkat cells. In conclusion, our data suggest that the DIP–DHA conjugate exhibits significantly more potent effects on CEM and Jurkat cells than that of DIP or DHA alone. These conjugates have potential use for treatment of patients with T cell acute lymphoblastic leukemia.
CXCL16 is a chemokine that is expressed in both transmembrane and secreted isoforms. Both variants have been implicated in atherosclerosis. Increased CXCL16 expression on the surface of human aortic ...smooth muscle cells induced by interferon gamma (IFNγ) signaling results in enhanced oxidized low density lipoprotein uptake and enhanced recruitment of pro-inflammatory cells. Docosahexaenoic acid (DHA), an omega-3 fatty acid, is known to inhibit IFNγ signaling in inflammatory cells. Therefore, we have investigated the effects of DHA treatment on the ability of IFNγ to induce CXCL16 expression in human aortic smooth muscle cells. We observed that DHA treatment significantly reduced IFNγ-induced CXCL16 expression. As a result, the pro-atherosclerotic functions of CXCL16 were also inhibited. Furthermore, IFNγ-induced STAT1 phosphorylation was inhibited by DHA, suggesting a potential mechanism. In conclusion, our data suggest inhibition of IFNγ signaling as one of the mechanisms behind the beneficial effects of DHA during atherosclerosis. These findings may prove to be important in other disease fields that identify IFNγ as a regulator.
The PIM kinases are a family of proteins recently identified as promising therapeutic targets in several cancers, including pancreatic, B-cell malignancies, acute leukemia, and prostate cancer among ...others. The family of PIM kinases is composed of three different members (PIM-1, -2, and -3) that are short-lived serine/threonine kinases involved in the regulation of a number of cellular pathways that are important for cancer cell growth and survival. The PIM kinases show high homology with each other, and exhibit functional redundancy in vitro and in vivo. Overexpression of PIM kinases promotes tumor growth through activation of several key cell-cycle progression and anti-apoptotic proteins, including BAD, p21, p27KIP, c-Myc, and AKT-1. Recently, overexpression of PIM-2 has been shown to have a protective effect against ultraviolet light induced DNA damage (Zirkin et al. J Biol Chem288:21770-83, 2013). We investigated the protective role of PIM kinases in chemotherapy-induced DNA damage, and whether inhibition of PIM kinases enhances anthracycline-induced DNA damage by inhibiting DNA repair, thus enhancing cell death in lymphoma cells lines.
Using immunobloting and RT-PCR, we found similarly low levels of PIM-1 and PIM-3, but a wide range of PIM-2 expression, in a panel of non-Hodgkin lymphoma (NHL) cell lines, including Raji, HS Sultan, Daudi, Farage, Granta519, and Toledo. Treatment of cells with doxorubicin (200-400 nM) resulted in up to a five fold increase gene transcription and expression of PIM-1 and PIM-2, which was maximal at 6 hours, and was associated with an increase in DNA damage as detected using acridine orange flow cytometry assay. We also tested the single agent effect of the pan-PIM kinase inhibitor, CX6258 on the cell lines. CX6258 alone inhibited cell growth in all NHL cell lines with varying degrees of potency with IC50ranging from 0.2 – 12.9 µM. The anti-cancer was associated most with PIM-2 expression, with the most sensitive cell lines, Daudi and Toledo, expressing the most PIM-2. Suppression of PIM-2 expression by shRNA significantly decreased proliferation, indicating that PIM-2 is a significant factor in cell growth. Treatment of NHL cells with CX6258 resulted in increased caspase-3 activation and PARP cleavage, decreased BAD phosphorylation, and apoptosis. Treatment with CX6258 also increased expression of p21, decreased expression of cyclins A1 and B1, and induced G2-M cell cycle arrest. The effect of combinations of CX6258 (5-50 µM) and doxorubicin (50-500 nM) on DNA damage and cell death was tested on HS sultan and Daudi cells. While doxorubicin alone resulted in a two-fold increase in DNA damage, this was significantly increased in the presence of CX6258 (12 fold). The addition of CX6258 inhibited the phosphorylation of the DNA repair proteins H2.AX, ATM, and Chk2 that occurred when the cells were treated with doxorubicin alone. The combination of CX6258 and doxorubicin was synergistic in inducing lymphoma cell death, with combination indexes ranging from 0.32-0.85.
Our findings suggest a mechanism for synergy where doxorubicin damages cellular DNA and initiates the DNA damage response, while CX6258 inhibits the upregulated PIM kinases from activating the proteins involved in the response. This synergistic anti-tumor activity is further strengthened by the CX6258 inhibition of cell cycle progression and anti-apoptotic proteins activated by the PIM kinases. Taken together, our results provide pre-clinical rationale for clinical testing of PIM kinase inhibitors in combination with doxorubicin in patients with NHL. It also suggests that CX6258 may similarly enhance the anti-cancer effects of other DNA damaging agents.
No relevant conflicts of interest to declare.
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