Involvement of Akt/NF‐κB pathway in N6‐isopentenyladenosine‐induced apoptosis in human breast cancer cells

N⁶‐isopentenyladenosine (i6A) inhibits the tumor cell growth by inducing cell apoptosis in various cancer cell lines. However, little is known regarding the mechanisms by which the drug induces cell apoptosis. In this study, we further explored the molecular mechanisms of i6A as an anticancer agent on a human breast cancer cell line MDA MB 231. Treatment with i6A decreased the cell proliferation of MDA MB 231 cells in a dose‐dependent manner by arresting the cells at G₀/G₁ phase. This effect was strongly associated with concomitant decrease in the level of cyclin D1, cyclin E, cdk2, and increase of p21waf1 and p27kip. In addition i6A also induced apoptotic cell death by increasing the expression of Bax, and decreasing the levels of Bcl‐2 and Bcl‐xL, and subsequently triggered mitochondria apoptotic pathway (release of cytochrome c and activation of caspase‐3). We observed that i6A suppressed the nuclear factor kappaB (NF‐κB) pathway and inhibited the Akt activation. The results of this study indicate that i6A decreases cell proliferation and induces apoptotic cell death in human breast cancer cells, possibly by decreasing signal transduction through the Akt/NF‐κB cell survival pathway. © 2010 Wiley‐Liss, Inc.     

The novel histone deacetylase inhibitor,AR‐42, inhibits gp130/Stat3 pathway and induces apoptosis and cell cycle arrest in multiple myeloma cells

Multiple myeloma (MM) remains incurable with current therapy, indicating the need for continued development of novel therapeutic agents. We evaluated the activity of a novel phenylbutyrate‐derived histone deacetylase inhibitor, AR‐42, in primary human myeloma cells and cell lines. AR‐42 was cytotoxic to MM cells at a mean LC₅₀ of 0.18 ± 0.06 μmol/l at 48 hr and induced apoptosis with cleavage of caspases 8, 9 and 3, with cell death largely prevented by caspase inhibition. AR‐42 downregulated the expression of gp130 and inhibited activation of STAT3, with minimal effects on the PI3K/Akt and MAPK pathways, indicating a predominant effect on the gp130/STAT‐3 pathway. AR‐42 also inhibited interleukin (IL)‐6‐induced STAT3 activation, which could not be overcome by exogenous IL‐6. AR‐42 also downregulated the expression of STAT3‐regulated targets, including Bcl‐xL and cyclin D1. Overexpression of Bcl‐xL by a lentivirus construct partly protected against cell death induced by AR‐42. The cyclin dependent kinase inhibitors, p16 and p21, were also significantly induced by AR‐42, which together with a decrease in cyclin D1, resulted in G₁ and G₂ cell cycle arrest. In conclusion, AR‐42 has potent cytotoxicity against MM cells mainly through gp130/STAT‐3 pathway. The results provide rationale for clinical investigation of AR‐42 in MM.     

Huaier aqueous extract inhibits proliferation of breast cancer cells by inducing apoptosis

Aqueous extract of Trametes robiniophila murr (Huaier) has been commonly used in China for cancer complementary therapy in recent years; however, the mechanisms of its anticancer effects are largely unknown. In the present study, we aim to investigate its inhibitory effect on both MCF‐7 and MDA‐MB‐231 cells, and explore the possible mechanisms of its anticancer effect. Cell viability and motility were measured by MTT and invasive assays, migration and scratch assays in vitro, respectively. The distribution of cell cycle, PI‐Annexin‐V staining and Rhodamine 123 assay were analyzed by flow cytometry, and western blot were used to test the apoptotic pathways. We found that Huaier extract could strongly inhibit cell viability of MCF‐7 and MDA‐MB‐231 cells in a time‐ and dose‐dependent manner; however, MDA‐MB‐231 cells showed more susceptibility to the treatment. Furthermore, cell invasiveness and migration were also suppressed with exposure to Huaier extract. We also indicated that Huaier could induce G0/G1 cell‐cycle arrest, p53 accumulation and activation selectively in MCF‐7 cells. Inspiringly, the PI‐Annexin‐V staining assay and western blot analysis confirmed cell apoptosis executed by caspase‐3. Decreased mitochondrial membrane potential by Rhodamine 123 assay and down‐regulation of Bcl‐2 and up‐regulation of BCL2‐associated X protein (BAX) indicated that Huaier induced apoptosis through the mitochondrial pathway. Caspase activation during Huaier‐induced apoptosis was confirmed by pan‐caspase inhibitor, Z‐VAD‐fmk. As expected, the inhibitor decreased Huaier‐induced apoptosis in both cell lines. Based on our findings, Huaier can induce cell apoptosis in both ER‐positive and ER‐negative breast cancer cell lines and is an effective complementary agent for breast cancer treatment. (Cancer Sci 2010; 101: 2375–2383)     

A novel human Fab antibody for Trop2 inhibits breast cancer growth in vitro and in vivo

Human trophoblastic cell surface antigen 2 (Trop2) has been suggested as an oncogene, which is associated with the different types of tumors. In this study, a human Fab antibody against Trop2 extracellular domain was isolated from phage library by phage display technology, and characterized by ELISA, FACS, fluorescence staining and Western blotting analysis. MTT, apoptosis assay and wound healing assay were employed to evaluate the inhibitory effects of Trop2 Fab on breast cancer cell growth in vitro, while tumor‐xenograft model was employed to evaluate the inhibitory effects on breast cancer growth in vivo. The results showed that Trop2 Fab inhibited the proliferation, induced the apoptosis and suspended the migration of MDA‐MB‐231 cells in a dose dependent manner. The expression caspase‐3 was activated, and the expression of Bcl‐2 was reduced while that of Bax was elevated in MDA‐MB‐231 cells by treating with Trop2 Fab. In addition, Trop2 Fab inhibited the growth of breast cancer xenografts and the expression of Bcl‐2 was reduced while that of Bax was elevated in xenografts. Trop2 Fab, which was isolated successfully in this research, is a promising therapeutic agent for the treatment of Trop2 expressing breast cancer.     

Anti‐proliferative and apoptosis‐inducing activity of lycopene against three subtypes of human breast cancer cell lines

Although lycopene, a major carotenoid component of tomatoes, has been suggested to attenuate the risk of breast cancer, the underlying preventive mechanism remains to be determined. Moreover, it is not known whether there are any differences in lycopene activity among different subtypes of human breast cancer cells. Using ER/PR positive MCF‐7, HER2‐positive SK‐BR‐3 and triple‐negative MDA‐MB‐468 cell lines, we investigated the cellular and molecular mechanism of the anticancer activity of lycopene. Lycopene treatment for 168 consecutive hours exhibited a time‐dependent and dose‐dependent anti‐proliferative activity against these cell lines by arresting the cell cycle at the G₀/G₁ phase at physiologically achievable concentrations found in human plasma. The greatest growth inhibition was observed in MDA‐MB‐468 where the sub‐G₀/G₁ apoptotic population was significantly increased, with demonstrable cleavage of PARP. Lycopene induced strong and sustained activation of the ERK1/2, with concomitant cyclin D1 suppression and p21 upregulation in these three cell lines. In triple negative cells, lycopene inhibited the phosphorylation of Akt and its downstream molecule mTOR, followed by subsequent upregulation of proapoptotic Bax without affecting anti‐apoptotic Bcl‐xL. Taken together, these data indicate that the predominant anticancer activity of lycopene in MDA‐MB‐468 cells suggests a potential role of lycopene for the prevention of triple negative breast cancer.     

Multiple antimelanoma potential of dry olive leaf extract

Various constituents of the olive tree (Olea europaea) have been traditionally used in the treatment of infection, inflammation, prevention of chronic diseases, cardiovascular disorders and cancer. The anticancer potential of dry olive leaf extract (DOLE) represents the net effect of multilevel interactions between different biologically active compounds from the extract, cancer cells and conventional therapy. In this context, it was of primary interest to evaluate the influence of DOLE on progression of the highly malignant, immuno‐ and chemoresistant type of skin cancer—melanoma. DOLE significantly inhibited proliferation and subsequently restricted clonogenicity of the B16 mouse melanoma cell line in vitro. Moreover, late phase tumor treatment with DOLE significantly reduced tumor volume in a syngeneic strain of mice. DOLE‐treated B16 cells were blocked in the G₀/G₁ phase of the cell cycle, underwent early apoptosis and died by late necrosis. At the molecular level, the dying process started as caspase dependent, but finalized as caspase independent. In concordance, overexpression of antiapoptotic members of the Bcl‐2 family, Bcl‐2 and Bcl‐XL, and diminished expression of their natural antagonists, Bim and p53, were observed. Despite molecular suppression of the proapoptotic process, DOLE successfully promoted cell death mainly through disruption of cell membrane integrity and late caspase‐independent fragmentation of genetic material. Taken together, the results of this study indicate that DOLE possesses strong antimelanoma potential. When DOLE was applied in combination with different chemotherapeutics, various outcomes, including synergy and antagonism, were observed. This requires caution in the use of the extract as a supplementary antitumor therapeutic.     

Naturally occurring asteriscunolide A induces apoptosis and activation of mitogen‐activated protein kinase pathway in human tumor cell lines

Sesquiterpene lactones have attracted much attention because they display a wide range of biological activities, including antitumor properties. Here, we show the effects of the naturally occurring sesquiterpene lactone asteriscunolide A (AS) on viability of human melanoma, leukemia and cells that overexpress antiapoptotic proteins, namely Bcl‐2 and Bcl‐x_L. All cell lines were sensitive to this compound, with IC₅₀ values of ～5 µM. The cytotoxic effects of AS were accompanied by a G₂‐M phase arrest of the cell cycle and a concentration‐ and time‐dependent appearance of apoptosis as determined by DNA fragmentation, translocation of phosphatidylserine to the cell surface and sub‐G₁ ratio. Apoptosis was associated with caspase‐3 activity and poly(ADP‐ribose) polymerase cleavage and was prevented by the nonspecific caspase inhibitor z‐VAD‐fmk, indicating that caspases are essential components in this pathway. The apoptotic effect of AS was also associated with (i) the release of cytochrome c from mitochondria which was accompanied by dissipation of the mitochondrial membrane potential (ΔΨ_m) and (ii) the activation of the mitogen‐activated protein kinases (MAPKs) pathway. AS‐induced cell death was potentiated by inhibition of extracellular signal‐regulated kinases (ERK) 1/2 signaling with U0126 and PD98059. Intracellular reactive oxygen species (ROS) seem to play a pivotal role in this process since high levels of ROS were produced early (1 h) and apoptosis was completely blocked by the free radical scavenger N‐acetyl‐L ‐cysteine (NAC). The present study demonstrates that AS‐induced cell death is mediated by an intrinsic‐dependent apoptotic event involving mitochondria and MAPKs, and through a mechanism dependent on ROS generation. © 2010 Wiley‐Liss, Inc.     

5,7,3′‐trihydroxy‐3,4′‐dimethoxyflavone‐induced cell death in human Leukemia cells is dependent on caspases and activates the MAPK pathway

Flavonoids are polyphenolic compounds which display a vast array of biological activities and are promising anticancer agents. In this study we investigated the effect of 5,7,3′‐trihydroxy‐3,4′‐dimethoxyflavone (THDF) on viability of nine human tumor cell lines and found that it was highly cytotoxic against leukemia cells. THDF induced G₂–M phase cell‐cycle arrest and apoptosis through a caspase‐dependent mechanism involving cytochrome c release, processing of multiple caspases (caspase‐3, ‐6, ‐7, and ‐9) and cleavage of poly(ADP‐ribose) polymerase. Overexpression of the protective mitochondrial proteins Bcl‐2 and Bcl‐x_L conferred partial resistance to THDF‐induced apoptosis. This flavonoid induced the phosphorylation of members of the mitogen‐activated protein kinases (MAPKs) family and cell death was attenuated by inhibition of c‐jun N‐terminal kinases/stress‐activated protein kinases (JNK/SAPK) and of extracellular signal‐regulated kinases (ERK) 1/2. In the present study we report that THDF‐induced cell death is mediated by an intrinsic dependent apoptotic event involving mitochondria and MAPKs, and through a mechanism independent of the generation of reactive oxygen species. The results suggest that THDF could be useful in the development of novel anticancer agents. Mol. Carcinog. © 2010 Wiley‐Liss, Inc.     

Inactivated Sendai virus particle upregulates cancer cell expression of intercellular adhesion molecule‐1 and enhances natural killer cell sensitivity on cancer cells

We have already reported that the inactivated Sendai virus (hemagglutinating virus of Japan; HVJ) envelope (HVJ‐E) has multiple anticancer effects, including induction of cancer‐selective cell death and activation of anticancer immunity. The HVJ‐E stimulates dendritic cells to produce cytokines and chemokines such as β‐interferon, interleukin‐6, chemokine (C‐C motif) ligand 5, and chemokine (C‐X‐C motif) ligand 10, which activate both CD8⁺ T cells and natural killer (NK) cells and recruit them to the tumor microenvironment. However, the effect of HVJ‐E on modulating the sensitivity of cancer cells to immune cell attack has yet to be investigated. In this study, we found that HVJ‐E induced the production of intercellular adhesion molecule‐1 (ICAM‐1, CD54), a ligand of lymphocyte function‐associated antigen 1, in several cancer cell lines through the activation of nuclear factor‐κB downstream of retinoic acid‐inducible gene I and the mitochondrial antiviral signaling pathway. The upregulation of ICAM‐1 on the surface of cancer cells increased the sensitivity of cancer cells to NK cells. Knocking out expression of ICAM‐1 in MDA‐MB‐231 cells using the CRISPR/Cas9 method significantly reduced the killing effect of NK cells on ICAM‐1‐depleted MDA‐MB‐231 cells. In addition, HVJ‐E suppressed tumor growth in MDA‐MB‐231 tumor‐bearing SCID mice, and the HVJ‐E antitumor effect was impaired when NK cells were depleted by treatment with the anti‐asialo GM1 antibody. Our findings suggest that HVJ‐E enhances NK cell sensitivity against cancer cells by increasing ICAM‐1 expression on the cancer cell surface.     

Reciprocal changes in gene expression profiles of cocultured breast epithelial cells and primary fibroblasts

The importance of epithelial‐stroma interaction in normal breast development and tumor progression has been recognized. To identify genes that were regulated by these reciprocal interactions, we cocultured a nonmalignant (MCF10A) and a breast cancer derived (MDA‐MB231) basal cell lines, with fibroblasts isolated from breast benign‐disease adjacent tissues (NAF) or with carcinoma‐associated fibroblasts (CAF), in a transwell system. Gene expression profiles of each coculture pair were compared with the correspondent monocultures, using a customized microarray. Contrariwise to large alterations in epithelial cells genomic profiles, fibroblasts were less affected. In MDA‐MB231 highly represented genes downregulated by CAF derived factors coded for proteins important for the specificity of vectorial transport between ER and golgi, possibly affecting cell polarity whereas the response of MCF10A comprised an induction of genes coding for stress responsive proteins, representing a prosurvival effect. While NAF downregulated genes encoding proteins associated to glycolipid and fatty acid biosynthesis in MDA‐MB231, potentially affecting membrane biogenesis, in MCF10A, genes critical for growth control and adhesion were altered. NAFs responded to coculture with MDA‐MB231 by a decrease in the expression of genes induced by TGFβ1 and associated to motility. However, there was little change in NAFs gene expression profile influenced by MCF10A. CAFs responded to the presence of both epithelial cells inducing genes implicated in cell proliferation. Our data indicate that interactions between breast fibroblasts and basal epithelial cells resulted in alterations in the genomic profiles of both cell types which may help to clarify some aspects of this heterotypic signaling. © 2009 UICC     

RNAi‐mediated CD73 suppression induces apoptosis and cell‐cycle arrest in human breast cancer cells

Ecto‐5′‐nucleotidase (CD73), a cell surface protein that hydrolyzes extracellular AMP into adenosine and phosphate, is overexpressed in many solid tumors. In this study, we tested the hypothesis that increased CD73 may promote tumor progression by examining the effect of CD73 suppression via RNA interference and CD73 overexpression on tumor growth in vivo and in vitro. Using digitized whole‐body images, plate clone forming assay and TUNEL assay in frozen tissue sections, we found that the cell growth rate was significantly lower in vivo and in vitro after CD73 suppression and late apoptosis was much higher in xenograft tumors developed from the CD73‐siRNA transfected MB‐MDA‐231 clone (P1). By flow cytometry, the P1 cell cycle was arrested in the G0/G1 phase. Moreover, Bcl‐2 was downregulated, while Bax and caspase‐3 were upregulated with CD73 suppression. CD73 inhibitor α,β‐methylene adenosine‐5′‐disphosphate (APCP) functioned similarly with RNAi‐mediated CD73 suppression. In addition, in transfected MCF‐7 cells, we found that CD73 overexpression increased cell viability and promoted cell cycle progression, depending on its enzyme activity. More intriguingly, CD73 overexpression in MCF‐7 breast cancer cells produces a tumorigenic phenotype. We conclude that CD73 plays an important role in breast cancer growth by affecting cell cycle progression and apoptosis. (Cancer Sci 2010; 101: 2561–2569)     

Antitumor effects of novel compound,guttiferone K,on colon cancer by p21Waf1/Cip1‐mediated G0/G1 cell cycle arrest and apoptosis

Low selectivity is one of the major problems of currently used anticancer drugs, therefore, there is a high demand for novel, selective antitumor agents. In this study, the anticancer effects and mechanisms of guttiferone K (GUTK), a novel polyprenylated acylphloroglucinol derivative isolated from Garcinia cowa Roxb., were examined for its development as a novel drug targeting colon cancer. GUTK concentration‐ and time‐dependently reduced the viability of human colon cancer HT‐29 cells (IC₅₀ value 5.39 ± 0.22 μM) without affecting the viability of normal human colon epithelial CCD 841 CoN cells and induced G₀/G₁ cell cycle arrest in HT‐29 cells by down‐regulating cyclins D1, D3 and cyclin‐dependent kinases 4 and 6, while selectively restoring p21Waf1/Cip1 and p27Kip1 to levels comparable to those observed in normal colon cells, without affecting their levels in normal cells. GUTK (10.0 μM) induced cleavage of PARP, caspases‐3, ‐8 and ‐9 and chromatin condensation to stimulate caspase‐3‐mediated apoptosis. The addition of a JNK inhibitor, SP600125, partially reversed GUTK‐induced caspase‐3 activity, indicating the possible involvement of JNK in GUTK‐induced apoptosis. Furthermore, GUTK (10 mg/kg, i.p.) significantly decreased the tumor volume in a syngeneic colon tumor model when used alone or in combination with 5‐fluorouracil without toxicity to the mice. Immunohistochemical staining of the tumor sections revealed a mechanism involving an increase in cleaved caspase‐3 and a decrease in cell proliferation marker Ki‐67. Our results support GUTK as a promising novel, potent and selective antitumor drug candidate for colon cancer.     

Development of DS‐5573a: A novel afucosylated mAb directed at B7‐H3 with potent antitumor activity

B7‐H3 is highly overexpressed in a variety of human clinical tumors, and its expression is significantly associated with poor outcomes. In our study, we aimed to develop new antitumor mAbs by employing cancer cell immunization, and succeeded in generating a mouse anti‐human B7‐H3 antibody (M30) that shows antitumor activity. M30 was humanized (Hu‐M30), and an afucosylated Hu‐M30 (DS‐5573a) was also generated. To assess the potency of DS‐5573a as a therapeutic mAb, we characterized this mAb and evaluated its antitumor activity in vitro and in vivo. Flow cytometry analysis showed that B7‐H3 proteins were expressed on various types of cancer cell lines broadly, and DS‐5573a binds to IgC1 and IgC2 domains of human B7‐H3. Antibody‐dependent cellular cytotoxicity activity of DS‐5573a was drastically enhanced against medium to high B7‐H3‐expressing cancer cell lines MDA‐MB‐231 and NCI‐H322. DS‐5573a also induced high antibody‐dependent cellular cytotoxicity activity against low B7‐H3‐expressing cancer cell line COLO205, whereas Hu‐M30 induced little activity against it. In addition, DS‐5573a was found to be a novel anti‐B7‐H3 antibody which showed antibody‐dependent cellular phagocytosis activity. Furthermore, DS‐5573a showed dose‐dependent and significant antitumor efficacy (0.03–3 mg/kg) in MDA‐MB‐231‐bearing SCID mice (which have functional natural killer cells and macrophages), but little antitumor efficacy in NOG mice (which lack natural killer cells and have reduced macrophage function). These results suggest that antitumor activity of DS‐5573a is mediated by effector cells, and this mAb could be a promising antitumor therapy for patients with a wide range of B7‐H3‐expressing tumors.     

PI(3,4)P2 plays critical roles in the regulation of focal adhesion dynamics of MDA‐MB‐231 breast cancer cells

Phosphoinositides play pivotal roles in the regulation of cancer cell phenotypes. Among them, phosphatidylinositol 3,4‐bisphosphate (PI(3,4)P₂) localizes to the invadopodia, and positively regulates tumor cell invasion. In this study, we examined the effect of PI(3,4)P₂ on focal adhesion dynamics in MDA‐MB‐231 basal breast cancer cells. Knockdown of SHIP2, a phosphatidylinositol 3,4,5‐trisphosphatase (PIP₃) 5‐phosphatase that generates PI(3,4)P₂, in MDA‐MB‐231 breast cancer cells, induced the development of focal adhesions and cell spreading, leading to the suppression of invasion. In contrast, knockdown of PTEN, a 3‐phosphatase that de‐phosphorylates PIP₃ and PI(3,4)P₂, induced cell shrinkage and increased cell invasion. Interestingly, additional knockdown of SHIP2 rescued these phenotypes. Overexpression of the TAPP1 PH domain, which binds to PI(3,4)P₂, and knockdown of Lpd, a downstream effector of PI(3,4)P₂, resulted in similar phenotypes to those induced by SHIP2 knockdown. Taken together, our results suggest that inhibition of PI(3,4)P₂ generation and/or downstream signaling could be useful for inhibiting breast cancer metastasis.