SW480 Human Colon Cancer Cells Research Articles

Lignans inhibit cell growth via regulation of Wnt/β-catenin signaling

As aberrant activation of Wnt/β-catenin signaling is one of the major mechanisms of carcinogenesis in colon cancer, identification of inhibitors of this pathway may aid in colon cancer prevention. We investigated the ability of the lignans arctiin, matairesinol and arctigenin from Saussurea salicifolia to inhibit Wnt/β-catenin signaling in SW480 human colon cancer cells. The lignans inhibited SW480 cell growth. In addition, the transcriptional activity of a reporter construct containing the TCF binding element (TBE) was decreased after the treatment with all three lignans. Although arctiin, matairesinol and arctigenin have similar structures, arctigenin affected Wnt/β-catenin signaling most significantly. Further, arctigenin reduced the level of β-catenin by inducing its phosphorylation and thus its degradation. Arctigenin also decreased expression of the β-catenin/TCF downstream genes CCND1, survivin and CTNNB1, and induced apoptosis. These results suggest that arctigenin, an aglycone with a methoxyl group, potently inhibits the growth of human colon cancer cells via the Wnt/β-catenin signaling pathway.

인체 대장암 세포주 SW480에서 docosahexaenoic acid에 의한 침윤억제 기전

대장암은 미국 등 서양 국가뿐만 아니라 국내에서도 2번째로 많이 발병이 되는 암으로 알려져 있다. 역학조사에 의하면 <TEX>${\omega}3$</TEX>-PUFAs를 많이 섭취한 인종에서 대장암 발생빈도가 감소하고 최근 <TEX>${\omega}3$</TEX>-PUFAs는 수종의 암에 대해 항암작용을 나타낸다고 한다. 이에 본 연구에서는 대장암에서 DHA 등 <TEX>${\omega}3$</TEX>-PUFA의 항침윤 기전을 규명하여 다음과 같은 결과를 얻었다. DHA및 EPA는 대장암 세포주 SW480의 증식을 농도 의존적으로 억제하였으나 AA는 거의 영향이 없었으며 TUNEL assay로 apoptotic cell death가 확인 되었다. DHA는 <TEX>$\beta$</TEX>-catenin 단백 및 TCF/LEF luciferase 활성을 농도 의존적으로 억제 하였다. SW480 세포의 침윤능은 DHA의 농도에 의존적으로 억제되었다. DHA처리 후 MMP-9 및 MMP-2 mRNA양이 감소되었을 뿐만 아니라 그 promoter의 reporter 활성도 억제되었다. NF-kB 및 p-IkB 단백질양도 DHA의 처리농도에 의존적으로 감소하였으며 NF-kB promoter의 활성도 억제되었다. 이상의 결과로 <TEX>${\omega}3$</TEX>-PUFA는 대장암에서 NF-kB 신호전달 차단에 의한 MMP-2 및 MMP-9 발현을 억제하여 침윤을 억제하여 항암작용을 나타낼 수 있음을 시사하며, 따라서 <TEX>${\omega}3$</TEX>-PUFA는 대장암의 예방 및 치료에 유용하게 사용될 수 있으리라 생각된다. Colon cancer is one of the most common malignancies in the western world and the second leading cause of cancer death in Korea. Epidemiology studies have shown a reduced incidence of colon cancer among populations consuming a large quantity of <TEX>${\omega}3$</TEX>-polyunsaturated fatty acids (<TEX>${\omega}3$</TEX>-PUFA) of marine origin. Recently, it has been found that <TEX>${\omega}3$</TEX>-PUFA has an antineoplastic effect in several cancers. This study was designed to investigate the mechanism of the anti-invasive effect of <TEX>${\omega}3$</TEX>-PUFA in colon cancer. <TEX>${\omega}3$</TEX>-PUFA, docosahexaenoic acids (DHA) and eicosapentaenoic acid (EPA) treatment resulted in a dose-dependent inhibition of cell growth in SW480 human colon cancer cells. In contrast, arachidonic acid (AA), a <TEX>${\omega}6$</TEX>-PUFA, exhibited no significant effect. This action likely involves apoptosis, given that DHA treatment increased apoptotic cells in TUNEL assay. Moreover, invasiveness of SW480 cells was inhibited following treatment of DHA in a dose-dependent manner; in contrast, AA had no effect. The levels of MMP-9 and MMP-2 mRNA decreased after DHA pretreatment. MMP-9 and MMP-2 promoter activities were also inhibited by DHA treatment. The levels of NF-kB and p-IkB protein were down-regulated by DHA pretreatment in a dose dependent manner. In addition, DHA inhibited NF-kB promoter reporter activities. These findings suggest that <TEX>${\omega}3$</TEX>-PUFA may inhibit cancer cell invasion by inhibition of MMPs via reduction of NF-kB in colon cancer. In conclusion, <TEX>${\omega}3$</TEX>-PUFA could be used for chemoprevention and treatment of human colon cancer.

Abstract 5646: Protein-bound polysaccharide from Phellinus linteus inhibits tumor growth, invasion and angiogenesis of SW480 human colon cancer cells by modulating Wnt/b-catenin signaling

Abstract It is known that polysaccharide extracted from Phellinus linteus (PL) mushroom possess antitumor activity. We previously have demonstrated that PL has direct antitumor effect through apoptosis and G2/M cell cycle blockade in SW480 human colon cancer cells. However, the molecular mechanisms responsible for antitumor and antiinvasive behavior of PL remain to be explored. Employing SW480 colon cancer cells which are overexpressed B-catenin, we demonstratd that PL significantly inhibited cellular proliferation and remarkable decrease of B-catenin expression on Western blot and indirect immunofluorescence detection in PL treated cells. Cyclin D1, one of the B-catenin controlled downstream genes and TCF/LEF transcription activity were also significantly reduced when PL was treated. PL inhibited in vitro invasion and motility of SW480 cells and the activity of matrix metalloproteinase (MMP)-9 on zymography. In addition, PL inhibited human umbilical vein endothelial cell (HUVEC) proliferation and capillary tube formation. Tumor growth of SW480 cells implanted into nude mice were significantly inhibited with PL treatment in a dose-dependent manner. Immunohistochemical analysis of xenografted tumor tissue showed that apoptotic index was markedly increased and nuclear staining of b-catenin was decreased. However, the proliferation index and microvessel density were significantly decreased. These data demonstrate that PL downregulates Wnt/b-catenin signaling pathway and inhibits multiple steps in tumor invasion in certain colon cancer cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5646.

Abstract 427: Targeted inhibition of the mTOR complex components, RAPTOR and RICTOR, attenuates progression of colorectal cancer

Abstract Colorectal cancer (CRC) is the second leading cause of cancer deaths in the USA. The mammalian target of rapamycin (mTOR) acts downstream of PI3K to regulate cell growth, proliferation and survival. The mTOR kinase nucleates two distinct complexes, mTORC1 and mTORC2. The mTOR-RAPTOR complex (mTORC1) regulates translation initiation through the effectors S6K and 4E-BP1, while the mTOR-RICTOR complex (mTORC2) regulates the actin cytoskeleton and activates Akt, a key regulator of cell survival. Rapamycin inhibits the kinase activity of mTORC1; prolonged rapamycin treatment also inhibits mTORC2 assembly and Akt activation in certain cells. The purpose of the present study was to determine the effect of targeting mTORC1 and mTORC2 upon progression of CRCs. METHODS. HCT116 and SW480 human colon cancer cells were treated with rapamycin. Cells were also transfected with shRNA directed against RAPTOR or RICTOR. Effects on migration (measured by wound healing assay) and invasion (measured by transwell matrigel assay) were analyzed. Changes in actin cytoskeleton and cell-cell contacts were determined by immunofluorescence microscopy. RESULTS. (i) Immunohistochemical analysis showed that the mTORC1 and mTORC2 components, mTOR, RAPTOR and RICTOR, are overexpressed in primary CRCs and matched liver metastases compared to normal colonic tissue. (ii) Treatment with rapamycin significantly decreased the migration and invasion of HCT116 and SW480 cells. (iii) Stable shRNA-mediated knockdown of RAPTOR and RICTOR significantly decreased the migration and invasion of HCT116 and SW480 cells. (iv) Stable shRNA-mediated knockdown of RAPTOR and RICTOR leads to mesenchymal-to-epithelial transition, as evidenced by increased E-cadherin and decreased vimentin levels. (v) Stable shRNA-mediated knockdown of RAPTOR and RICTOR leads to characteristic rearrangements in actin cytoskeleton as well as increased cell-cell contact. CONCLUSIONS. The mTORC1 and mTORC2 components, mTOR, RAPTOR and RICTOR, are overexpressed in both primary CRCs as well as matched liver metastases. Although the mTOR signaling axis is primarily associated with growth of normal and cancer cells, we demonstrate that stable inhibition of both mTORC1 and mTORC2 decreases migration and invasion capabilities of CRC cells as well as causing a mesenchymal-to-epithelial change in phenotype. The mechanism for this decrease in migration and invasion may be via modulation of actin cytoskeletal rearrangement and cell-cell contact. Thus, targeted inhibition of mTOR signaling represents a novel therapeutic strategy to attenuate the progression and metastasis of CRCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 427.

Abstract 5684: B-catenin-dependent and independent changes in gene expression induced by sphingolipids

Abstract Dietary supplementation with sphingolipids inhibits colon tumorigenesis in carcinogen-treated CF1 mice and in multiple intestinal neoplasia (Min) mice that produce intestinal tumors spontaneously. In both cancer models the regulation of β-catenin by sphingolipid metabolites is an important early event. Stabilization and nuclear translocation of β-catenin and its interaction with T-cell factor (TCF)/lymphoid enhancer factor (LEF) play important roles to activate the transcription of genes associated with cancer development and progression. Sphingosine and ceramides, major digestion products of complex sphingolipids in the diet, down-regulate β-catenin transcriptional activity in a dose- and time-dependent manner. Our data indicate that sphingosine and ceramides suppress SW480 human colon cancer cell growth in a concentration-dependent manner. In the present study, we investigated the dependency of sphingosine- and ceramide-induced changes in gene expression on β-catenin transcriptional activity by comparing control and β-catenin siRNA treated cells. We observed changes in the expression of genes involved in signal transduction, transcription factors, cell cycle control and DNA damage repair, cell adhesion, cell invasion and metastasis, angiogenesis, and apoptosis and cell senescence. Most genes were regulated by all metabolites; however, some genes were only targeted by specific metabolites. Furthermore, only a small number of genes regulated by the sphingolipid metabolites relied completely on the activation of the β-catenin transcriptional activity. Further experiments are currently being performed to confirm the function of β-catenin on the regulation of these genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5684.

Recombinant human endostatin inhibited the lymphangiogenesis and lymph node metastasis in a mouse xenograft model of colon cancer

Objective To observe the effects of recombinant human endostatin, Endostar, on tumor lymphangiogenesis and lymph node metastasis in a mouse xenograft model of colon cancer. Methods Human colon cancer SW620 cells were implanted subcutaneously into the hind flank of the male nu/nu mice to estab-lish a xenograft model. The control group was treated with normal saline, and the Endostar group was treated with Endostar by subcutaneous injection every other day. Results (1) The rate of lymph node metastasis was 10% (1/10) in the Endostar group, and 80% (8/10) in the control group (P <0.01 ) ; (2) The den-sity of lymphatic vessels in the Endostar group was decreased significantly as compared with the control group (7.17 ± 0. 75 vs. 14.83 ± 0.98 per field, P < 0.05 ) ; (3) Endostar inhibited the expression of vascular endo-thehal cell growth factor (VEGF)-C and VEGF-D proteins in tumor tissues and SW620 cells. Conclusion Recombinant human endostatin suppressed lymphangiogenesis and lymph node metastasis by inhibiting the ex-pression of VEGF-C and VEGF-D in a mouse xenograft model of colon cancer. Key words: Endostatin; Colorectal carcinoma; Lymphatic vessel; Metastasis

Cucurbitacin B induces G2 arrest and apoptosis via a reactive oxygen species‐dependent mechanism in human colon adenocarcinoma SW480 cells

Cucurbitacin B (cucB) is a triterpenoid constituent of Cucurbitaceae vegetables and a promising phytochemical for cancer prevention. However, the mechanism of anti-tumor activity of cucB remains unknown, especially in colon cancers. Here, we demonstrate for the first time that cucB inhibited growth of human colon cancer SW480 cells through a reactive oxygen species (ROS)-dependent mechanism. CucB induced G(2) phase arrest and apoptosis in a dose-dependent manner. At the molecular level, cucB reduced the expression of cyclin B1 and cdc25C proteins and activated caspases in SW480 cells. On the other hand, the state of phosphorylation of signaling transducer and activator of transcription 3 (STAT3) was unchanged. We found that cucB increased intracellular ROS levels, and N-acetylcysteine, a well-known antioxidant, reduced the changes in expression of the molecules, and suppressed both G(2) arrest and apoptosis. These results suggested that cucB induced G(2) arrest and apoptosis through a STAT3-independent but ROS-dependent mechanism in SW480 cells.

Benzophenones and Biflavonoids from Garcinia livingstonei Fruits

A series of 13 known compounds, including seven benzophenones [guttiferone A (1), guttiferone K (2), xanthochymol (3), guttiferone E (4), cycloxanthochymol (5), isoxanthochymol (6), and gambogenone (7)], five biflavonoids [amentoflavone (8), 3,8''-biapigenin (9), (+)-volkensiflavone (10), (+)-morelloflavone (11), and (+)-fukugiside (12)], and the xanthone derivative alloathyriol (13), were identified from the fruits of Garcinia livingstonei (Clusiaceae). This is the first time that compounds 2-7, 9, 12, and 13 have been reported in this species. The cytotoxicity of benzophenones 1 and 2 was assessed for their effect on HCT-116, HT-29, and SW-480 human colon cancer cell lines. Both compounds exhibited strong activity against HCT-116 and HT-29 cell lines with IC(50) values between 5 and 10 microM, and somewhat weaker activity with SW-480 cells (IC(50) values ranging from 18 to 25 microM).

Substrate stiffness and the receptor-type tyrosine-protein phosphatase alpha regulate spreading of colon cancer cells through cytoskeletal contractility

Microenvironmental clues are critical to cell behavior. One of the key elements of migration is the generation and response to forces. Up to now, there is no definitive concept on how the generation and responses to cellular forces influence cancer-cell behavior. Here, we show that expression of receptor-type tyrosine-protein phosphatase alpha (RPTPalpha) in human SW480 colon cancer cells sets a threshold for the response to matrix forces by changing cellular contractility. This can be explained as an RPTPalpha-mediated increase in contractility with a consecutive increase in number and size of adhesion sites and stress fibers. These effects are mediated through myosin light chain kinase and largely independent of Rho/Rho-kinase (ROCK) signaling. In addition, we report that RPTPalpha influences spreading on low-rigidity surfaces, binding of collagen-coated beads and expression of RPTPalpha is required for invasion into the chorioallantoic membrane. These data suggest that force-responsive proteins such as RPTPalpha can influence cancer-cell behavior and identify potential targets for cancer therapy.

Anticancer effect of Lycium barbarum polysaccharides on colon cancer cells involves G0/G1 phase arrest

Colorectal cancer is one of the most common cancers worldwide. The anticancer effect of Wolfberry (Lycium barbarum) polysaccharide (LBP) on colon cancer cells is largely unknown. To investigate the growth effect of LBP on human colon cancer cell and its possible mechanisms, human colon cancer SW480 and Caco-2 cells were treated with 100-1,000 mg/l LBP for 1-8 days. Cell growth was measured by MTT assay and crystal violet assay. Distribution of the cell cycle was analyzed by flow cytometry. Western blotting was used to indicate changes in the level of cyclins and cyclin-dependent kinases (CDKs). LBP treatment inhibited both colon cancer cell lines in a dose-dependent manner. At concentrations from 400 to 1,000 mg/l, LBP significantly inhibited the growth of SW480 cells (400 mg/l, P < 0.01; 800 and 1,000 mg/l, P < 0.001); while at concentrations from 200 to 1,000 mg/l, LBP significantly inhibited the growth of Caco-2 cells (200 mg/l, P < 0.05; 400-1,000 mg/l, P < 0.001). Crystal violet assay showed that LBP had a long-term anti-proliferative effect. More importantly, cells were arrested at the G0/G1 phase. The changes in cell-cycle-associated protein, cyclins, and CDKs were consistent with the changes in cell-cycle distribution. This is one of the first studies to focus on LBP-induced interruption of the cell cycle in human colon carcinoma cells. The results suggest that LBP is a candidate anticancer agent.

SiRNA-mediated IGF-1R inhibition sensitizes human colon cancer SW480 cells to radiation

Purpose. Insulin like growth factor receptor 1 (IGF-1R) is well-documented to play a key role in radiation response and tumor radiosensitivity, thus offering an attractive clinic drug target to enhance tumor sensitivity to anti-cancer radiotherapy. Material and methods. Human colon carcinoma SW480 cells were transfected with the specific small interference RNA (siRNA) expression vector (pkD-shRNA-IGF-1R-V2) designed to target IGF-1R mRNA. The expression of IGF-1R mRNA and its protein among the transfected and untransfected cells were detected by semi-quantitative RT-PCR and ELISA assay. The changes in cell radiosensitivity were examined by MTT assay. Results. Transfection of mammalian expression vector pkD containing IGF-1R siRNA was shown to reduce IGF-1R mRNA levels by up to 95%. ELISA assay detected a similar inhibition of IGF-1R protein levels in cells transfected with IGF-1R siRNA. SW480 cells transfected with the expression vector for siRNA significantly rendered cells more sensitive to radiation and the highest radiation enhancement ratio was 2.02 ± 0.08. Conclusion. These data provide the first evidence that specific siRNA fragment (pkD-shRNA-IGF-1R-V2) targeting human IGF-1R mRNA is able to enhance colon cancer radiosensitivity. Also results indicated that, combining IGF-1R siRNA and radiation significantly enhances antitumor efficacy compared with either modality alone.

Abstract B15: Targeted inhibition of mTOR signaling attenuates the growth and progression of colorectal cancer

Abstract Introduction: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the USA. The mammalian target of rapamycin (mTOR) acts downstream of PI3K to regulate cell growth, proliferation and survival. The mTOR kinase nucleates two distinct complexes, mTORC1 and mTORC2. The mTOR-RAPTOR complex (mTORC1) regulates translation initiation through the effectors S6K and 4E-BP1, while the mTOR-RICTOR-mSin1 complex (mTORC2) regulates the actin cytoskeleton and activates Akt, a key regulator of cell survival. Rapamycin inhibits the kinase activity of mTORC1; prolonged rapamycin treatment also inhibits mTORC2 assembly and Akt activation in certain cells. Furthermore, rapamycin mediated mTORC1 inhibition leads to loss of mTOR-S6K-IRS1 dependent feedback inhibition, thereby resulting in increased Akt activation. The purpose of the present study was to determine: (i) sensitivity of CRC cells to rapamycin treatment, and (ii) effect of targeting mTORC1 and mTORC2 upon CRC proliferation, apoptosis, cell cycle progression, migration, invasion and xenograft growth. Methods: HCT116, KM20, Caco-2 and SW480 human colon cancer cells were treated with rapamycin. Cells were also transfected with siRNA/shRNA directed against mTOR, RAPTOR or RICTOR. Effects on cell proliferation (measured by Coulter counter cell counts), apoptosis (measured by detecting the level of histone-associated DNA fragments), cell cycle progression (measured by fluorescence-activated cell sorting), migration (measured by wound healing assay), invasion (measured by transwell matrigel assay) and subcutaneous growth were analyzed. Results: (i) Immunohistochemical analysis showed that the mTORC1 and mTORC2 components, mTOR, RAPTOR and RICTOR are overexpressed in primary CRCs and matched liver metastases compared to normal colonic tissue. (ii) Treatment with rapamycin significantly decreased the proliferation of HCT116 and KM20 cells (rapamycin sensitive); however, rapamycin did not alter SW480 or Caco-2 proliferation (rapamycin resistant). (iii) Treatment with rapamycin significantly decreased the migration and invasion of HCT116 and SW480 cells. (iv) Transient siRNA-mediated knockdown of RAPTOR decreased proliferation of KM20 cells only, while knockdown of RICTOR decreased proliferation of all four cell lines. (v) Stable shRNA-mediated knockdown of mTORC1 and mTORC2 components leads to decreased proliferation, increased apoptosis and inhibition of G1-S phase cell cycle progression in HCT116 cells. (vi) Stable shRNA-mediated knockdown of mTORC1 and mTORC2 leads to decreased proliferation, but only mTORC2 knockdown leads to increased apoptosis in SW480 cells. (vii) Targeted inhibition of mTORC1 and mTORC2 attenuates growth of rapamycin-sensitive and — resistant CRCs in xenografted nude mice. Conclusions: The mTORC1 and mTORC2 components, mTOR, RAPTOR and RICTOR are overexpressed in CRCs. Transient inhibition of mTORC2, but not mTORC1, decreased the proliferation of rapamycin-sensitive and —resistant CRC cells. Stable inhibition of mTORC2 decreased proliferation and induced apoptosis in rapamycin-sensitive and —resistant CRC cells. Finally, targeted inhibition of both mTORC1 and mTORC2 attenuates subcutaneous growth of CRCs in vivo. Thus, inhibition of mTORC2 activity represents a novel therapeutic strategy for treatment of rapamycin-sensitive and, more importantly, rapamycin-resistant CRCs. Citation Information: Cancer Res 2009;69(23 Suppl):B15.

Novel nitro-oxy derivatives of celecoxib for the regulation of colon cancer cell growth

Celecoxib is a non-steroidal anti-inflammatory drug (NSAID) developed as a selective inhibitor of cyclooxygenase-2 (COX-2). Despite the associated cardiovascular toxicity risk, celecoxib has been found to be effective in reducing cancer risk in animal and human studies. In the present study the antiproliferative activity of novel nitro-oxy-methyl substituted analogues of celecoxib (NO-cel), potentially less cardiotoxic, has been investigated in vitro on human colon cancer cells and compared with action of the parent drug. Moreover, experiments were performed in order to evaluate whether COX-2 pharmacological inhibition may affect β-catenin/E-cadherin signalling pathway. All the tested analogues of celecoxib exerted a significant antiproliferative activity on COX-2 positive HT-29 human colon cancer cells, being less effective on the COX-2 negative SW-480 human colon cancer cell line. In particular, the analogue displaying two nitro-oxy functions fully mimicked the known inhibitory properties of celecoxib, including inhibition of COX-2, as well as of ERK/MAPK and β-catenin signalling pathways. Interestingly, the latter compound also elicited a strong reorganization of the β-catenin/E-cadherin complex, which has been suggested to be relevant for colon carcinogenesis. On these premises, NO-cel analogues of celecoxib can represent promising colon cancer chemopreventive agents potentially able to affect colon cancer development.

Cancer Prevention of Anthocyanins in the Purple Sweetpotato bred in Kansas

Anthocyanins are versatile and plentiful flavonoid pigments that account for the purple color in may plant foods such as red grapes, berries, red cabbages, and purple sweetpotato. Although anthocyanins have been epidemiologically associated with reduced cancer risk, the anticancer ability of the purple sweetpotato has not been well investigated. Considering the high contents of anthocyanin in the purple sweetpotato, we have bred purple‐fleshed sweetpotato cultivars using seeds from controlled crosses provided by the International Potato Center in Lima, Peru. Over 2000 seedlings from four full‐sib progenies cultured at the John C. Pair Horticulture Research Center, Kansas, have been segregated by multiple traits based upon flesh pigmentation and fiber contents. We and determined the prominent anthocyanin contents by HPLC‐MS, including cyanidin 3‐sophoroside‐5‐glucoside (m/z 774), p‐hydroxybenzoylated cyanidin 3‐sophoroside‐5‐glucoside (m/z 894), caffeoylated cyanidin 3‐sophoroside‐5‐glucoside (m/z 908), and peonidin 3‐(6,6′‐caffeoyl‐p‐hydroxybenzoylsophoroside)‐5‐glucoside (m/z 1055). Furthermore, the selected purple sweetpotato extracts with high levels of anthocyanins have been assessed in the human colon cancer SW480 cells for potential anticancer mechanisms. Better understanding of the underlying mechanisms may provide scientific evidence and health benefits of purple sweetpotato grown in Kansas.

Asiatic Acid Induces Colon Cancer Cell Growth Inhibition and Apoptosis through Mitochondrial Death Cascade

Cancer is one of the leading causes of death in the world. The triterpenoid compound asiatic acid derived from the tropical medicinal plant Centella asiatica displays cytotoxic activity on fibroblast cells and several other kinds of cells. The present work studies asiatic acid-mediated growth inhibition of cancer cells and the underlying mechanism. Asiatic acid markedly inhibited cancer cell proliferation. Apoptosis of SW480 human colon cancer cells was induced by asiatic acid as shown by flow cytometry, DNA fragmentation and nuclear chromatin condensation experiments. Through increasing mitochondrial membrane permeability and cytochrome c release from mitochondria into cytosol, asiatic acid induced caspase-9 activity, which further activated caspase-3 and poly(ADP-ribose) polymerase cleavage resulting in irreversible apoptotic death in the tumor cells. Taken together, these results suggest that mitochondrial death apoptosis cascade plays very important roles in asiatic acid-induced cancer apoptosis.

Curcumin Inhibits the Proteasome Activity in Human Colon Cancer Cells In vitro and In vivo

Curcumin (diferuloylmethane) is the major active ingredient of turmeric (Curcuma longa) used in South Asian cuisine for centuries. Curcumin has been shown to inhibit the growth of transformed cells and to have a number of potential molecular targets. However, the essential molecular targets of curcumin under physiologic conditions have not been completely defined. Herein, we report that the tumor cellular proteasome is most likely an important target of curcumin. Nucleophilic susceptibility and in silico docking studies show that both carbonyl carbons of the curcumin molecule are highly susceptible to a nucleophilic attack by the hydroxyl group of the NH(2)-terminal threonine of the proteasomal chymotrypsin-like (CT-like) subunit. Consistently, curcumin potently inhibits the CT-like activity of a purified rabbit 20S proteasome (IC(50) = 1.85 micromol/L) and cellular 26S proteasome. Furthermore, inhibition of proteasome activity by curcumin in human colon cancer HCT-116 and SW480 cell lines leads to accumulation of ubiquitinated proteins and several proteasome target proteins, and subsequent induction of apoptosis. Furthermore, treatment of HCT-116 colon tumor-bearing ICR SCID mice with curcumin resulted in decreased tumor growth, associated with proteasome inhibition, proliferation suppression, and apoptosis induction in tumor tissues. Our study shows that proteasome inhibition could be one of the mechanisms for the chemopreventive and/or therapeutic roles of curcumin in human colon cancer. Based on its ability to inhibit the proteasome and induce apoptosis in both HCT-116 and metastatic SW480 colon cancer cell lines, our study suggests that curcumin could potentially be used for treatment of both early-stage and late-stage/refractory colon cancer.

Kaempferol sensitizes colon cancer cells to TRAIL-induced apoptosis

Kaempferol is a natural compound contained in edible plants, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent. Here, we show for the first time that the combined treatment with kaempferol and TRAIL drastically induced apoptosis in human colon cancer SW480 cells, compared to single treatments. Kaempferol markedly up-regulated TRAIL receptors, DR5 and DR4. DR5 but not DR4 siRNA efficiently blocked apoptosis induced by the co-treatment with kaempferol and TRAIL, indicating that DR5 up-regulation by kaempferol helps to enhance TRAIL actions. Moreover, we examined the combined effect on normal human cells. The co-treatment induced no apoptosis in normal human peripheral blood mononuclear cells and little apoptosis in normal human hepatocytes. These results suggest that kaempferol is useful for TRAIL-based treatments for cancer.

Hyperosmotic stress up-regulates the expression of major vault protein in SW620 human colon cancer cells

The major vault protein (MVP) is the major constituent of the vault particle, the largest ribonuclear protein complex described to date and is identical to lung resistance-related protein (LRP). Although MVP is also expressed in several normal tissues, little is known about its physiological role. MVP played a protective role against some xenobiotics and other stresses. We thus investigated the effect of osmotic stress on MVP expression by treating human colon cancer SW620 cells with sucrose or NaCl. The expression level of both MVP protein and MVP mRNA was increased by the osmostress. Sucrose or sodium chloride could also enhance MVP promoter activity. Inhibition of p38 MAPK in SW620 cells by SB203580 inhibited the expression of MVP under hyperosmotic stress. These findings suggested that osmotic stress up-regulated the MVP expression through p38 MAPK pathway. Down-regulation of MVP expression by MVP interfering RNA (RNAi) in SW620 cells increased the sensitivity of the cells to hyperosmotic stress and enhanced apoptosis. Furthermore, MVP RNAi prevented the osmotic stress-induced, time-dependent increase in phosphorylated Akt. These findings suggest that the PI3K/Akt pathway might be implicated in the cytoprotective effect of MVP.Our data demonstrate that exposure of cells to hyperosmotic stress induces MVP that might play an important role in the protection of the cells from the adverse effects of osmotic stress.

Activation of Protein Kinase G Increases the Expression of p21CIP1, p27KIP1, and Histidine Triad Protein 1 through Sp1

The anticancer role of cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase G (PKG) has become of considerable interest, but the underlying mechanisms are not fully established. In this study, we examined the effects of activation of PKG on the expression of three tumor suppressor proteins in human SW480 colon cancer cells. Our results revealed that treatment with cell permeable cGMP derivatives, or the cGMP phosphodiesterase inhibitor sulindac sulfone (exisulind, aptosyn, hereafter called exisulind) led to increased expression of the tumor suppressor proteins p21(CIP1), p27(KIP1), and Histidine triad protein 1 (HINT1), and their corresponding mRNAs. Overexpression of PKG Ibeta also caused increased expression of the p21(CIP1), p27(KIP1), and HINT1 proteins. Both the p21(CIP1) and p27(KIP1) promoters contain Sp1 binding sites and they were activated by PKG in luciferase reporter assays. Specific Sp1 sites in the p21 and p27 promoters were sufficient to mediate PKG-induced luciferase reporter activity, suggesting an interaction between Sp1 and PKG. Indeed, we found that PKG can phosphorylate Sp1 on serine residue(s) and this resulted in transcriptional activation of Sp1. Knockdown of Sp1 expression with siRNA inhibited the increased expression of p21(CIP1), p27(KIP1), and HINT1 induced by the cGMP derivative 8-pCPT-cGMP in SW480 cells. These novel effects of PKG activation on the expression of three tumor suppressor genes may explain, at least in part, the anticancer effects of activation of PKG. They also provide a rationale for further developing activators of PKG for the prevention and treatment of cancer.

IPP5, a Novel Protein Inhibitor of Protein Phosphatase 1, Promotes G1/S Progression in a Thr-40-dependent Manner

Protein phosphatase 1 (PP1) is a major serine/threonine phosphatase that controls gene expression and cell cycle progression. Here we describe the characterization of a novel inhibitory molecule for PP1, human inhibitor-5 of protein phosphatase 1 (IPP5). We find that IPP5, containing the PP1 inhibitory subunits, specifically interacts with the PP1 catalytic subunit and inhibits PP1 phosphatase activity. Furthermore, the mutation of Thr-40 within the inhibitory subunit of IPP5 into Ala eliminates the phosphorylation of IPP5 by protein kinase A and its inhibitor activity to PP1, whereas the mutation of Thr-40 within a truncated form of IPP5 into Asp can serve as a dominant active form of IPP5 in inhibiting PP1 activity. In IPP5-negative SW480 and IPP5-highly positive SW620 human colon cancer cells, we find that overexpression of IPP5 promotes the growth and accelerates the G(1)-S transition of SW480 cells in a Thr-40-dependent manner, which could be reversed by downregulation of the PP1 expression. Moreover, silencing of IPP5 inhibits the growth of SW620 cells both in vitro and in nude mice possibly by inducing G(0)/G(1) arrest but not by promoting apoptosis. According to its role in the promotion of cell cycle progression and cell growth, IPP5 up-regulates the expression of cyclin E and the phosphorylated form of retinoblastoma protein. Our findings suggest that IPP5, by acting as an inhibitory molecule for PP1, can promote tumor cell growth and cell cycle progression, and may be a promising target in cancer therapeutics in IPP5-highly expressing tumor cells.