Anna L. Greenshields

Curcumin-induced apoptosis in ovarian carcinoma cells is p53-independent and involves p38 mitogen-activated protein kinase activation and downregulation of Bcl-2 and survivin expression and Akt signaling†

New cytotoxic agents are urgently needed for the treatment of advanced ovarian cancer because of the poor long‐term response of this disease to conventional chemotherapy. Curcumin, obtained from the rhizome of Curcuma longa, has potent anticancer activity; however, the mechanism of curcumin‐induced cytotoxicity in ovarian cancer cells remains a mystery. In this study we show that curcumin exhibited time‐ and dose‐dependent cytotoxicity against monolayer cultures of ovarian carcinoma cell lines with differing p53 status (wild‐type p53: HEY, OVCA429; mutant p53: OCC1; null p53: SKOV3). In addition, p53 knockdown or p53 inhibition did not diminish curcumin killing of HEY cells, confirming p53‐independent cytotoxicity. Curcumin also killed OVCA429, and SKOV3 cells grown as multicellular spheroids. Nuclear condensation and fragmentation, as well as DNA fragmentation and poly (ADP‐ribose) polymerase‐1 cleavage in curcumin‐treated HEY cells, indicated cell death by apoptosis. Procaspase‐3, procaspase‐8, and procaspase‐9 cleavage, in addition to cytochrome c release and Bid cleavage into truncated Bid, revealed that curcumin activated both the extrinsic and intrinsic pathways of apoptosis. Bax expression was unchanged but Bcl‐2, survivin, phosphorylated Akt (on serine 473), and total Akt were downregulated in curcumin‐treated HEY cells. Curcumin also activated p38 mitogen‐activated protein kinase (MAPK) without altering extracellular signal‐regulated kinase 1/2 activity. We conclude that p53‐independent curcumin‐induced apoptosis in ovarian carcinoma cells involves p38 MAPK activation, ablation of prosurvival Akt signaling, and reduced expression of the antiapoptotic proteins Bcl‐2 and survivin. These data provide a mechanistic rationale for the potential use of curcumin in the treatment of ovarian cancer.

Curcumin causes superoxide anion production and p53-independent apoptosis in human colon cancer cells

Curcumin from the rhizome of theCurcuma longa plant has chemopreventative activity and inhibits the growth of neoplastic cells. Since p53 has been suggested to be important for anticancer activity by curcumin, we investigated curcumin-induced cytotoxicity in cultures of p53(+/+) and p53(-/-) HCT-116 colon cancer cells, as well as mutant p53 HT-29 colon cancer cells. Curcumin killed wild-type p53 HCT-116 cells and mutant p53 HT-29 cells in a dose- and time-dependent manner. In addition, curcumin-treated p53(+/+) HCT-116 cells and mutant p53 HT-29 cells showed upregulation of total and activated p53, as well as increased expression of p53-regulated p21, PUMA (p53 upregulated modulator of apoptosis), and Bax; however, an equivalent cytotoxic effect by curcumin was observed in p53(+/+) and p53(-/-) HCT-116 cells, demonstrating that curcumin-induced cytotoxicity was independent of p53 status. Similar results were obtained when the cytotoxic effect of curcumin was assessed in wild-type p53 HCT-116 cells after siRNA-mediated p53 knockdown. Chromatin condensation, poly (ADP-ribose) polymerase-1 cleavage and reduced pro-caspase-3 levels in curcumin-treated p53(+/+) and p53(-/-) HCT-116 cells suggested that curcumin caused apoptosis. In addition, exposure to curcumin resulted in superoxide anion production and phosphorylation of oxidative stress proteins in p53(+/+) and p53(-/-) HCT-116 cells. Collectively, our results indicate that, despite p53 upregulation and activation, curcumin-induced apoptosis in colon cancer cells was independent of p53 status and involved oxidative stress. Curcumin may therefore have therapeutic potential in the management of colon cancer, especially in tumorsthatare resistant to conventional chemotherapydue todefects inp53 expression or function.

Piperine inhibits the growth and motility of triple-negative breast cancer cells

Piperine, an alkaloid from black pepper, is reported to have anticancer activities. In this study, we investigated the effect of piperine on the growth and motility of triple-negative breast cancer (TNBC) cells. Piperine inhibited the in vitro growth of TNBC cells, as well as hormone-dependent breast cancer cells, without affecting normal mammary epithelial cell growth. Exposure to piperine decreased the percentage of TNBC cells in the G2 phase of the cell cycle. In addition, G1- and G2-associated protein expression was decreased and p21(Waf1/Cip1) expression was increased in piperine-treated TNBC cells. Piperine also inhibited survival-promoting Akt activation in TNBC cells and caused caspase-dependent apoptosis via the mitochondrial pathway. Interestingly, combined treatment with piperine and γ radiation was more cytotoxic for TNBC cells than γ radiation alone. The in vitro migration of piperine-treated TNBC cells was impaired and expression of matrix metalloproteinase-2 and -9 mRNA was decreased, suggesting an antimetastatic effect by piperine. Finally, intratumoral administration of piperine inhibited the growth of TNBC xenografts in immune-deficient mice. Taken together, these findings suggest that piperine may be useful in the treatment of TNBC.

Thymoquinone, A Bioactive Component of Black Caraway Seeds, Causes G1 Phase Cell Cycle Arrest and Apoptosis in Triple-Negative Breast Cancer Cells with Mutant p53

Thymoquinone (TQ) from black caraway seeds has several anticancer activities; however, its effect on triple-negative breast cancer (TNBC) cells that lack functional tumor suppressor p53 is not known. Here, we explored the growth inhibitory effect of TQ on 2 TNBC cell lines with mutant p53. Cell metabolism assays showed that TQ inhibited TNBC cell growth without affecting normal cell growth. Flow cytometric analyses of TQ-treated TNBC cells showed G1 phase cell cycle arrest and apoptosis characterized by the loss of mitochondrial membrane integrity. Western blots of lysates from TQ-treated TNBC cells showed cytochrome c and apoptosis-inducing factor in the cytoplasm, as well as caspase-9 activation consistent with the mitochondrial pathway of apoptosis. Caspase-8 was also activated in TQ-treated TNBC cells, although the mechanism of activation is not clear at this time. Importantly, TQ-induced apoptosis was only partially inhibited by zVAD-fmk, indicating a role for caspase-independent effector molecules. Poly(ADP-ribose) polymerase cleavage and increased γH2AX, as well as reduced Akt phosphorylation and decreased expression of X-linked inhibitor of apoptosis, were evident in TQ-treated cells. Finally, TQ enhanced cisplatin- and docetaxel-induced cytotoxicity. These findings suggest that TQ could be useful in the management of TNBC, even when functional p53 is absent.

The Dietary Flavonoid Fisetin Causes Cell Cycle Arrest, Caspase‐Dependent Apoptosis, and Enhanced Cytotoxicity of Chemotherapeutic Drugs in Triple‐Negative Breast Cancer Cells

Fisetin (3,3′,4′,7‐tetrahydroxyflavone), a flavonoid found in a number of fruits and vegetables, has diverse biological activities, including cytotoxic effects on cancer cells. In this study, we investigated the effect of fisetin on triple‐negative breast cancer (TNBC) cells. TNBC has a poorer prognosis than other types of breast cancer and treatment options for this disease are limited. Fisetin inhibited the growth of MDA‐MB‐468 and MDA‐MB‐231 TNBC cells, as well as their ability to form colonies, without substantially affecting the growth of non‐malignant cells. In addition, fisetin inhibited the growth of estrogen receptor‐bearing MCF‐7 breast cancer cells and human epidermal growth factor receptor 2‐overexpressing SK‐BR‐3 breast cancer cells. Fisetin inhibited TNBC cell division and induced apoptosis, which was associated with mitochondrial membrane permeabilization and the activation of caspase‐9 and caspase‐8, as well as the cleavage of poly(ADP‐ribose) polymerase‐1. Induction of caspase‐dependent apoptosis by fisetin was confirmed by reduced killing of TNBC cells in the presence of the pan‐caspase inhibitors Z‐VAD‐FMK and BOC‐D‐FMK. Decreased phosphorylation of histone H3 at serine 10 in fisetin‐treated TNBC cells at G2/M phase of the cell cycle suggested that fisetin‐induced apoptosis was the result of Aurora B kinase inhibition. Interestingly, the cytotoxic effect of cisplatin, 5‐fluorouracil, and 4‐hydroxycyclophosphamide metabolite of cyclophosphamide on TNBC cells was increased in the presence of fisetin. These findings suggest that further investigation of fisetin is warranted for possible use in the management of TNBC. J. Cell. Biochem. 117: 1913–1925, 2016.

Contribution of reactive oxygen species to ovarian cancer cell growth arrest and killing by the anti-malarial drug artesunate.

Ovarian cancer is a leading cause of cancer‐related death in women and the most lethal gynecological malignancy in the developed world. The morbidity and mortality of ovarian cancer underscore the need for novel treatment options. Artesunate (ART) is a well‐tolerated anti‐malarial drug that also has anti‐cancer activity. In this study, we show that ART inhibited the in vitro growth of a panel of ovarian cancer cell lines, as well as the growth of ovarian cancer cells isolated from patients. Moreover, ART decreased tumor growth in vivo in a mouse model of ovarian cancer. ART‐treated ovarian cancer cells showed a strong induction of reactive oxygen species (ROS) and reduced proliferation. ROS‐dependent cell cycle arrest occurred in the G2/M phase whereas ROS‐independent cell cycle arrest occurred in the G1 phase, depending on the concentration of ART to which ovarian cancer cells were exposed. The anti‐proliferative effect of ART was associated with altered expression of several key cell cycle regulatory proteins, including cyclin D3, E2F‐1, and p21, as well as inhibition of mechanistic target of rapamycin signaling. Exposure of ovarian cancer cells to higher concentrations of ART resulted in ROS‐dependent DNA damage and cell death. Pretreatment of ovarian cancer cells with a pan‐caspase inhibitor or ferroptosis inhibitor decreased but did not completely eliminate ART‐mediated cytotoxicity, suggesting the involvement of both caspase‐dependent and caspase‐independent pathways of killing. These data show that ART has potent anti‐proliferative and cytotoxic effects on ovarian cancer cells, and may therefore be useful in the treatment of ovarian cancer.

Inhibitory effect of iron withdrawal by chelation on the growth of human and murine mammary carcinoma and fibrosarcoma cells

Since iron uptake is essential for cell growth, rapidly dividing cancer cells are sensitive to iron depletion. To explore the effect of iron withdrawal on cancer cell growth, mouse and human mammary carcinoma cells (4T1 and MDA-MB-468, respectively) and mouse and human fibrosarcoma cells (L929 and HT1080, respectively) were cultured in the absence or presence of DIBI, a novel iron-chelating polymer containing hydroxypyridinone iron-ligand functionality. Cell growth was measured by a colorimetric assay for cell metabolic activity. DIBI-treated 4T1, MDA-MB-468, L929 and HT1080 cells, as well as their normal counterparts, showed a dose- and time-dependent reduction in growth that was selective for human cancer cells and mouse fibrosarcoma cells. The inhibitory effect of DIBI on fibrosarcoma and mammary carcinoma cell growth was reversed by addition of exogenous iron in the form of iron (III) citrate, confirming the iron selectivity of DIBI and that its inhibitory activity was iron-related. Fibrosarcoma and mammary carcinoma cell growth inhibition by DIBI was associated with S-phase cell cycle arrest and low to moderate levels of cell death by apoptosis. Consistent with apoptosis induction following DIBI-mediated iron withdrawal, fibrosarcoma and mammary carcinoma cells exhibited mitochondrial membrane permeabilization. A comparison of DIBI to other iron chelators showed that DIBI was superior to deferiprone and similar to or better than deferoxamine for inhibition of fibrosarcoma and mammary carcinoma cell growth. These findings suggest that iron withdrawal from the tumor microenvironment with a selective and potent iron chelator such as DIBI may prevent or inhibit tumor progression.

Resveratrol, piperine and apigenin differ in their NADPH-oxidase inhibitory and reactive oxygen species-scavenging properties

BACKGROUND Many plant-derived chemicals have been studied for their potential benefits in ailments including inflammation, cancer, neurodegeneration, and cardiovascular disease. The health benefits of phytochemicals are often attributed to the targeting of reactive oxygen species (ROS). However, it is not always clear whether these agents act directly as antioxidants to remove ROS, or whether they act indirectly by blocking ROS production by enzymes such as NADPH oxidase (NOX) enzymes, or by influencing the expression of cellular pro- and anti- oxidants. HYPOTHESIS/PURPOSE Here we evaluate the pro- and anti-oxidant and NOX-inhibiting qualities of four phytochemicals: celastrol, resveratrol, apigenin, and piperine. STUDY DESIGN This work was done using the H661 cell line expressing little or no NOX, modified H661 cells expressing NOX1 and its subunits, and an EBV-transformed B-lymphoblastoid cell line expressing endogenous NOX2. ROS were measured using Amplex Red and nitroblue tetrazolium assays. In addition, direct ROS scavenging of hydrogen peroxide or superoxide generated were measured using Amplex Red and methyl cypridina luciferin analog (MCLA). RESULTS Of the four plant-derived compounds evaluated, only celastrol displayed NOX inhibitory activities, while celastrol and resveratrol both displayed ROS scavenging activity. Very little impact on ROS was observed with apigenin, or piperine. CONCLUSION The results of this study reveal the differences that exist between cell-free and intracellular pro-oxidant and antioxidant activities of several plant-derived compounds.

Piperine blocks interleukin-2-driven cell cycle progression in CTLL-2 T lymphocytes by inhibiting multiple signal transduction pathways.

Piperine, a pungent alkaloid found in the fruits of black pepper plants, has diverse physiological effects, including the ability to inhibit immune cell-mediated inflammation. Since the cytokine interleukin-2 (IL-2) is essential for the clonal expansion and differentiation of T lymphocytes, we investigated the effect of piperine on IL-2 signaling in IL-2-dependent mouse CTLL-2 T lymphocytes. Tritiated-thymidine incorporation assays and flow cytometric analysis of Oregon Green 488-stained cells showed that piperine inhibited IL-2-driven T lymphocyte proliferation; however, piperine did not cause T lymphocytes to die or decrease their expression of the high affinity IL-2 receptor, as determined by flow cytometry. Western blot analysis showed that piperine blocked the IL-2-induced phosphorylation of signal transducer and activator of transcription (STAT) 3 and STAT5 without affecting the upstream phosphorylation of Janus kinase (JAK) 1 and JAK3. In addition, piperine inhibited the IL-2-induced phosphorylation of extracellular signal-regulated kinase 1/2 and Akt, which are signaling molecules that regulate cell cycle progression. Piperine also suppressed the expression of cyclin-dependent kinase (Cdk) 1, Cdk4, Cdk6, cyclin B, cyclin D2, and Cdc25c protein phosphatase by IL-2-stimulated T lymphocytes, indicating G0/G1 and G2/M cell cycle arrest. Piperine-mediated inhibition of IL-2 signaling and cell cycle progression in CTLL-2 T lymphocytes suggests that piperine should be further investigated in animal models as a possible natural source treatment for T lymphocyte-mediated transplant rejection and autoimmune disease.

4T1 Murine Mammary Carcinoma Cells Enhance Macrophage-Mediated Innate Inflammatory Responses.

Tumor progression and the immune response are intricately linked. While it is known that cancers alter macrophage inflammatory responses to promote tumor progression, little is known regarding how cancers affect macrophage-dependent innate host defense. In this study, murine bone-marrow-derived macrophages (BMDM) were exposed to murine carcinoma-conditioned media prior to assessment of the macrophage inflammatory response. BMDMs exposed to 4T1 mammary carcinoma-conditioned medium demonstrated enhanced production of pro-inflammatory cytokines tumor necrosis factor α, interleukin-6, and CCL2 in response to lipopolysaccharide (LPS) while production of interleukin-10 remained unchanged. The increased LPS-induced production of pro-inflammatory cytokines was transient and correlated with enhanced cytokine production in response to other Toll-like receptor agonists, including peptidoglycan and flagellin. In addition, 4T1-conditioned BMDMs exhibited strengthened LPS-induced nitric oxide production and enhanced phagocytosis of Escherichia coli. 4T1-mediated augmentation of macrophage responses to LPS was partially dependent on the NFκB pathway, macrophage-colony stimulating factor, and actin polymerization, as well as the presence of 4T1-secreted extracellular vesicles. Furthermore, peritoneal macrophages obtained from 4T1 tumor-bearing mice displayed enhanced pro-inflammatory cytokine production in response to LPS. These results suggest that uptake of 4T1-secreted factors and actin-mediated ingestion of 4T1-secreted exosomes by macrophages cause a transient enhancement of innate inflammatory responses. Mammary carcinoma-mediated regulation of innate immunity may have significant implications for our understanding of host defense and cancer progression.