Mechanism Of Cell Growth Inhibition Research Articles

Functional Screen for Secreted Proteins by Monoclonal Antibody Library and Identification of Mac-2 Binding Protein (Mac-2BP) as a Potential Therapeutic Target and Biomarker for Lung Cancer

Identification of secreted proteins of lung cancer could provide new candidates of serum biomarkers for cancer diagnosis or targets for therapeutic intervention. In this study, we developed a novel strategy that combined functional monoclonal antibody library screening technique and mass spectrometry to identify functional secreted proteins. BALB/c mice were immunized with cancer cells isolated from fresh human lung cancer tissues. The monoclonal antibody library containing 1160 mAbs was established with the mouse spleen cells, whose serum had most anti-proliferative effect on lung cancer cells. Monoclonal antibodies were subjected to an immunoreactive and functional screen and monoclonal antibodies that reacted strongly with secreted proteins in condition medium and lung cancer tissues with high inhibotion of cell proliferation were selected. Antigens that recognized by antibodies were obtained by immunoprecipitation and then identified by mass spectrometry. Mac-2-binding protein (Mac-2BP), the antigen of 13H3 antibody, was identified using this approach. Functional studies demonstrated that the 13H3 antibody suppressed lung cancer cell lines ANIP-973 and A549 proliferation in vitro and inhibit ANIP973 xenograft tumors growth in vivo by inducing cell-cycle arrest at G1 phase, with up-regulation of p27 and down-regulation of cyclin D1. Moreover, the serum level of Mac-2BP was significantly higher in lung cancer patients than healthy controls. At a cutoff value of 6 μg/ml, Mac-2BP might be a diagnostic biomarker of lung cancer, especially for SCLC. Mac-2BP concentrations of 6 μg/ml or higher was associated with poor overall survival in univariate analysis, and was an independent predictor in the multivariate COX analysis. Together, these results firstly demonstrated that Mac-2BP can be used as a therapeutic target and potential biomarker for lung cancer. Our strategy is feasible, which may facilitate the identification of novel secreted biomarkers of lung cancer.

Proteomic Analysis of Covalent Modifications of Tubulins by Isothiocyanates1–3

Although isothiocyanates (ITC), which are found in cruciferous vegetables, have been shown to inhibit carcinogenesis in animal models and induce apoptosis and cell cycle arrest in tumor cells, the biochemical mechanisms of cell growth inhibition by these compounds are not fully understood. Studies have reported that ITC binding to intracellular proteins may be an important event for initiating apoptosis. Specific protein target(s) and molecular mechanisms for ITC have been investigated in human lung cancer A549 cells using proteomic tools. Cells were treated with various amounts (1–100μmol/L) of radiolabeled phenethyl-ITC (PEITC) and sulforaphane (SFN) and the extracted proteins resolved using 2-dimensional gel electrophoresis. The results of mass spectrometric analyses suggested that tubulin may be an in vivo binding target for ITC. The binding of ITC to tubulin was associated with growth arrest. The proliferation of A549 cells was significantly reduced by ITC, with benzyl-ITC (BITC) having a greater relative activity than PEITC or SFN. Mitotic arrest and apoptosis as well as disruption of microtubule polymerization were induced in the order: BITC > PEITC > SFN. An analysis of tubulins isolated from BITC-treated A549 cells showed that Cys347, a conserved cysteine in all α-tubulin isoforms, was covalently modified by BITC. Taken together, these results suggest that tubulin is a binding target of ITC and that this interaction can lead to growth inhibition and apoptosis.

Screening of SirT1 activating compounds and their cytotoxicity in prostate cancer cell lines.

e13545 Background: Our previous studies demonstrated that the human androgen receptor (AR) is an acetylated protein, and acetylation of the AR in the hinge region is essential for its ligand-dependent transactivation. The AR acetylation mimic mutant, ARK630Q, and a somatic mutation from the prostate cancer patient at the AR acetylating motif, ARK630T, showed enhanced receptor/coactivator binding and led to accelerated prostate cancer cell growth. SirT1, an NAD+-dependent class III HDAC, suppresses AR and inhibits prostate cell proliferation via deacetylation of AR. Deletion of the SirT1 gene in mice resulted in prostatic intraepithelial neoplasia (PIN) lesion formation associated with reduced autophagy. Our hypothesis is that small molecules with SirT1 activating properties could be potential novel agents for treatment of AR-expressing prostate cancer. Methods: We performed in-silico screening for small molecule antagonists of SirT1 based on computational model prediction of a known SirT1 activator, resveratrol, and identified potential activators for SirT1. The cytotoxicity of these compounds to the prostate cancer cell lines was tested by MTT assay. Results: Among the twelve compounds tested, three compounds, C5, C6 and C10, were found to inhibit AR expression in LNCaP cells and have cytotoxicity in several prostate cell lines including LNCaP, C4W2 and NeuT-transformed prostate epithelial cell line at concentration of 50 µM. Conclusions: Currently, we are testing the effects of these small molecules on the NAD+-dependent deacetylation activity of SirT1 and their effects on AR acetylation as its molecular mechanism of cell growth inhibition. The ultimate goal of this study is to develop early phase clinical trials in prostate cancer patients by blocking AR acetylation using these SirT1 activators.

Cytostatic effect of xanthone-loaded mPEG-b-p(HPMAm-Lac2) micelles towards doxorubicin sensitive and resistant cancer cells

Xanthone exhibits several medicinal activities and especially it inhibits the growth of cancer cells. However, the use of xanthone is limited because of its low aqueous solubility and systemic toxicity. In the present study xanthone was loaded into poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-dilactate] mPEG-b-p(HPMAm-Lac2) micelles in order to overcome these drawbacks. It was shown that xanthone could be loaded in these micelles up to 2mg/mL with ∼100% entrapment efficiency and ∼20% loading capacity. The average particle diameter of the xanthone loaded mPEG-b-p(HPMAm-Lac2) micelles as determined by dynamic light scattering ranged from 84 to 112nm. In vitro assays showed that xanthone in its free form as well as loaded in polymeric micelles had a high cytotoxicity towards both doxorubicin sensitive and, importantly, resistant cancer cells. On the other hand empty mPEG-b-p(HPMAm-Lac2) micelles did not show any cytotoxicity towards normal cells (PBMCs). Interestingly, the cytostatic effect of xanthone towards normal cells was masked when loaded in the micelles. The mechanism of cell growth inhibition by xanthone-loaded polymeric micelles was mediated through induction of apoptosis, as evidenced from a subdiploid peak of propidium iodide stained cells using flow cytometric analysis. From the results of this study it can be concluded that xanthone has potent anticancer activity not only on sensitive but also on doxorubicin resistant cancer cell lines. mPEG-b-p(HPMAm-Lac2) micelles are therefore attractive delivery systems of xanthone for the treatment of cancer.

Molecular targeting therapy with angiotensin II receptor blocker for prostatic cancer

Angiotensin II (Ang-II) plays a key role as a vasoconstrictor in controlling blood pressure and electrolyte/fluid homeostasis. Recently it has also been shown that this peptide is a cytokine, acting as a growth factor in cardiovascular and stromal cells. In addition, the physiological function of Ang-II seems to be similar in prostate cancer and stromal cells. It is widely assumed that Ang-II facilitates the growth of both cells, and its receptor blockers (ARBs) have the potential to inhibit the growth of various cancer cells and tumors through the Ang-II receptor type 1 (AT1 receptor). The mechanism of cell growth inhibition by ARBs has been considered to be that of suppression of the signal transduction systems activated by growth factors or cytokines in prostate cancer cells, and suppression of angiogenesis. This review highlights the possible use of ARBs as novel agents for prostatic diseases including prostate cancer and benign hypertrophy, and covers related literature.

Chamaejasmine Induces Apoptosis in Human Lung Adenocarcinoma A549 Cells through a Ros-Mediated Mitochondrial Pathway

In the present study, the anticancer activity of chamaejasmine towards A549 human lung adenocarcinoma cells was investigated. In order to explore the underlying mechanism of cell growth inhibition of chamaejasmine, cell cycle distribution, ROS generation, mitochondrial membrane potential (Δψm) disruption, and expression of cytochrome c, Bax, Bcl-2, caspase-3, caspase-9 and PARP were measured in A549 cells. Chamaejasmine inhibited the growth of A549 cells in a time and dose-dependent manner. The IC50 value was 7.72 µM after 72 h treatment. Chamaejasmine arrested the cell cycle in the G2/M phase and induced apoptosis via a ROS-mediated mitochondria-dependent pathway. Western blot analysis showed that chamaejasmine inhibited Bcl-2 expression and induced Bax expression to desintegrate the outer mitochondrial membrane and causing cytochrome c release. Mitochondrial cytochrome c release was associated with the activation of caspase-9 and caspase-3 cascade, and active-caspase-3 was involved in PARP cleavage. All of these signal transduction pathways are involved in initiating apoptosis. To the best of our knowledge, this is the first report demonstrating the cytotoxic activity of chamaejasmine towards A549 in vitro.

Anti-proliferative effects of the novel squamosamide derivative (FLZ) on HepG2 human hepatoma cells by regulating the cell cycle-related proteins are associated with decreased Ca 2+/ROS levels

FLZ is a synthetic novel squamosamide derivative and has previously been proved to be a potential drug for Parkinson’s disease and Alzheimer’s disease. FLZ has strong antioxidant activity, which implies that FLZ could eliminate excessive intracellular reactive oxygen species (ROS) in tumor cells and induce a pathway related to low cellular ROS levels, thereby inhibiting tumor cells proliferation. However, few reports have focused on the antitumor effects of FLZ. In this study, we investigated the antitumor efficacy of FLZ in HepG2 cells and the mechanism of cell growth inhibition. FLZ effectively inhibited HepG2 cell proliferation in a dose- and time-dependent manner; meanwhile, it was minimally toxic to normal cells. FLZ induced a significant decrease in oxidative stress through elimination of excessive intracellular ROS and strengthening of the glutathione antioxidant system. In addition, FLZ can effectively attenuate redundant [Ca 2+] i, thereby avoiding uncontrolled amplification by Ca 2+/ROS positive feedback. Furthermore, Western blot showed that FLZ inhibited phosphorylation of Akt and retinoblastoma protein (Rb), down-regulated the expressions of cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2), and enhanced the expression of CDK inhibitor p27 kip1, while did not affect CDK4 expression. These results suggest that FLZ has potent anti-proliferative activity against malignant human hepatoma cells via modulation of the expression or activation of cell-cycle regulatory proteins, which are associated with decreased Ca 2+/ROS levels, and indicate that FLZ is a potential liver cancer drug worthy of further research and development.

Abstract 5576: Rhamnetin inhibits human prostate cancer cell growth through cell-cycle arrest by modulating expression and function of key cell-cycle regulators and survival molecules

Abstract Development of effective agents for treatment of prostate cancer has become a national medical priority. Rhamnetin (3,5,3,4-tetrahydroxy-7-methoxyflavone), a natural compound known as O-methylated flavonol, abundantly present in cloves, sweet annie, annual wormwood, green vegetables and orchard grass has shown to possess anti-inflammatory and antioxidant properties. However, the anticancer properties of rhamnetin have not been fully elucidated. In the present study we demonstrated the molecular mechanism of cell growth inhibition and induction of apoptosis in human prostate carcinoma LNCaP and PC-3 cells by rhamnetin. Treatment of these cells with rhamnetin resulted in a dose (5, 10, 20, 40 microM) and time (24, 48 and 72 h) dependent inhibition of growth, G2-S phase arrest of the cell cycle and DNA fragmentation. This effect was associated with a marked decrease in the protein expression of cyclin D1, D2, and E and their activating partner, cyclin-dependent kinase (cdk) 2, 4, and 6, with concomitant upregulation of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18. Rhamnetin treatment also resulted in alteration in Bax/Bcl2 ratio in favor of apoptosis, which was associated with the release of cytochrome-c and induction of apoptotic protease-activating factor-1 (Apaf-1). This effect was found to result in a significant increase in cleaved fragments of caspase-9, -3, and poly (ADP-ribose) polymerase (PARP). Further, rhamnetin treatment resulted in down modulation of XIAP, survivin, and c-Myc protein expressions. Taken together, we concluded that molecular mechanisms during rhamnetin-mediated growth inhibition and induction of apoptosis in both androgen-responsive, LNCaP and androgen-refractory, PC-3 cells was due to modulation in cell-cycle machinery, disruption of mitochondrial function, and inhibition of cell survival machinery. For the first time we provide evidence that rhamnetin acts on potential molecular targets of cell cycle regulation and survival to elicit anticancer effects in prostate cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5576. doi:10.1158/1538-7445.AM2011-5576

Abstract P5-06-11: Knockdown of Splicing Factor SRp20 Results in Cell Line-Dependent Apoptosis in Breast Cancer Cells

Abstract Background: Cancer-specific alternative splicing as well as aberrant splicing factor expression in tumors have been reported (Korner M et al, Am. J. Pathol. 175:461-472, 2009). Splicing factor SRp20 belongs to a family of highly conserved serine-arginine-rich proteins with multiple functions in RNA processing such as spliceosome assembly and alternative splicing (Graveley BR, RNA. 6(9):1197-211, 2000). Materials and Methods: We established stable MDA-MB-231, MCF-7, and T47D cell lines carrying doxycycline (Doxy)-inducible SRp20shRNA and assessed Doxy-induced knockdown (KD) of SRp20 by western blot. We performed cell growth curves in triplicate using the MTT assay to determine the effect of SRp20 KD in these cell lines after Doxy induction of SRp20shRNA, and compared these to controls. For apoptosis assays, we stained SRp20shRNA sublines with Hoechst 33342 and counted apoptotic cells under the microscope. We also carried out western blots to examine the expressions of Bcl-2 and cleaved caspases-3, -6, -7, and -9. Results and Discussion: We found that the expression of SRp20 was upregulated in immortalized human mammary epithelial cells (HMECs), compared to controls, and that the level of SRp20 correlated with the transformation state. Knockdown of SRp20 was seen to be 88%, 58%, and 52% in MDA-MB-231, MCF-7, and T47D cells, respectively. However, SRp20 KD suppressed breast cancer cell growth in a cell line-dependent manner. Cell growth after 7 days was decreased in estrogen receptor-negative (ER-) MDA-MB-231 cells (91.4%), and estrogen receptor-positive (ER+) MCF-7 (73.2%), while SRp20 KD had no effect on growth of ER+ T47D cells. The differences between MCF-7 and T47D were notable, since SRp20 was suppressed by -50% in both lines. We found that the number of apoptotic cells in MDA-MB-231 line remarkably increased after Doxy treatment, indicating that the inhibition of cell growth was due to apoptosis. Further, immunoblot analysis of Bcl-2 and cleaved caspases in MDA-MB-231 cells indicated that the SRp20 KD activates the intrinsic apoptotic pathway in these breast cancer cells. We are presently assessing the expressions of these proteins in MCF-7 and T47D cells and investigating the mechanistic basis for this cell line-dependent effect of SRp20 KD. In summary, we report here a novel finding that inhibition of splicing factor SRp20 inhibits cell growth in some breast cancer cell lines. The intrinsic apoptotic pathway appears to be the main mechanism of cell growth inhibition in these cells. Our results indicate that SRp20 plays a major role in the survival of some breast cancer cell lines. This splicing factor may represent a novel therapeutic target for the treatment of some breast cancers. (Supported in part by CA40570 and CA138762 from NCI to WTB, in part by IDPH to XH, and in part by UIC) Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-06-11.

Shikonin inhibits the proliferation and induces the apoptosis of human HepG2 cells

This study investigated the potential of shikonin as an anticancer agent against liver cancer and an in vitro human hepatoma cancer model system. The HepG2 cell line was the hepatoma cancer model in the present study. The inhibitory effect of shikonin on the growth of HepG2 cells was measured by MTT assay. To explore the underlying mechanism of cell growth inhibition of shikonin, the cell cycle distribution, DNA fragmentation, mitochondrial membrane potential (ΔΨm) disruption, and expression of Bax and Bcl-2 were measured in HepG2 cells. The activity of shikonin in inducing apoptosis was investigated through the detection of Annexin V signal and CD95 expression by flow cytometry and electron microscopy, respectively. Shikonin inhibited the growth of HepG2 cells in a dose-dependent manner. The IC50 value (inhibiting cell growth by 50%) was 4.30 mg/mL. Shikonin inhibited cell growth in a dose-dependent manner and blocked HepG2 cell cycle progression at the S phase. The changes in mitochondrial morphology, dose-dependently decreased in ΔΨm, were observed in different concentrations of the drug treatment group. Western blot analysis showed that cajanol inhibited Bcl-2 expression and induced Bax expression. Furthermore, we show that shikonin increases Annexin V signal and CD95 (Fas/APO) expression, resulting in apoptotic cell death of HepG2 cells. In addition, lump formation of intranuclear chromatin, pyknosis of cell nucleus, deletion of microvillus, vacuolar degeneration of mitochondria, reduction of rough endoplasmic reticulum, and resolution of free ribosome, etc., associated with apoptosis were discovered by electron microscopy in HepG2 cells after 48 h treatment. Shikonin inhibited HepG2 cells, possibly through the pathway of inducing early apoptosis, and was beneficial for restoring the apoptotic sensitivity of HepG2 cells by CD95, and should therefore be considered as a candidate agent for the prevention or treatment of human hepatoma.

Fabrication of a β-TCP Nanomaterial and its Inhibitory Effects on Human Ovarian Cancer SKOV-3 Cells

Nanosized β-tricalcium phosphate (TCP) material was produced in this study using a wet precipitation method and characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD). Human ovarian sarcoma SKOV-3 cells were cultured and the influence of nanoscale β-TCP particles on SKOV-3 cell behavior was studied in vitro. As a result, β-TCP nanoparticles with average size of 100 nm were obtained. Cell growth of SKOV-3 cells was noticeably declined in the presence of β-TCP nanoparticles (200ng/ml). The distribution of cell cycle for SKOV-3 cells cultured with and without β-TCP nanomaterials was quite different. In G1 phase of cell cycle, the percentage of SKOV-3 cells cultured in the absence of β-TCP nanoparticles was significantly lower than that cultured in the presence of β-TCP nanoparticles (p<0.01). In S phase of cell cycle, on the other hand, the percentage of SKOV-3 cells cultured without β-TCP nanoparticles was noticeably increased compared with that cultured with β-TCP nanoparticles (p<0.01). Moreover, the expression of proliferating cell nuclear antigen (PCNA) in SKOV-3 cells cultured in medium containing 200ng/ml β-TCP nanopaticles was significantly lower than that in the cells cultured without β-TCP nanoparticles (p<0.01). In conclusion, the nanoscale β-TCP material synthesized in this study can exert anti-tumor effects on SKOV-3 cells through mechanisms of cell growth inhibition, downregulation of PCNA expression and cell cycle arrest at G1 phase.

Cajanol, a novel anticancer agent from Pigeonpea [ Cajanus cajan (L.) Millsp.] roots, induces apoptosis in human breast cancer cells through a ROS-mediated mitochondrial pathway

Cajanol (5-hydroxy-3-(4-hydroxy-2-methoxyphenyl)-7-methoxychroman-4-one) is an isoflavanone from Pigeonpea [ Cajanus cajan (L.) Millsp.] roots. As the most effective phytoalexin in pigeonpea, the cytotoxic activity of cajanol towards cancer cells has not been report as yet. In the present study, the anticancer activity of cajanol towards MCF-7 human breast cancer cells was investigated. In order to explore the underlying mechanism of cell growth inhibition of cajanol, cell cycle distribution, DNA fragmentation assay and morphological assessment of nuclear change, ROS generation, mitochondrial membrane potential (Δ Ψm) disruption, and expression of caspase-3 and caspase-9, Bax, Bcl-2, PARP and cytochrome c were measured in MCF-7 cells. Cajanol inhibited the growth of MCF-7 cells in a time and dose-dependent manner. The IC 50 value was 54.05 μM after 72 h treatment, 58.32 μM after 48 h; and 83.42 μM after 24 h. Cajanol arrested the cell cycle in the G2/M phase and induced apoptosis via a ROS-mediated mitochondria-dependent pathway. Western blot analysis showed that cajanol inhibited Bcl-2 expression and induced Bax expression to desintegrate the outer mitochondrial membrane and causing cytochrome c release. Mitochondrial cytochrome c release was associated with the activation of caspase-9 and caspase-3 cascade, and active-caspase-3 was involved in PARP cleavage. All of these signal transduction pathways are involved in initiating apoptosis. To the best of our knowledge, this is the first report demonstrating the cytotoxic activity of cajanol towards cancer cells in vitro.

Cytotoxic Activity of Isoliquiritigenin towards CCRF‐CEM Leukemia Cells and its Effect on DNA Damage

The cytotoxic activity of isoliquiritigenin (ISL) towards CCRF-CEM human T-cell leukemia cells and DNA damage induction were investigated in the present study. The anti-proliferative effect of ISL on CCRF-CEM cell line IN VITRO was analyzed. In order to further explore the underlying mechanism of cell growth inhibition of ISL towards CCRF-CEM cell line, the cell cycle distribution and mitochondrial membrane potential (DeltaPsim) disruption were measured. ISL exerted significant activity towards CCRF-CEM cells. The growth inhibitory ratio was concentration- and time-dependent with an IC(50) value of 18.38 microM. The cell cycle was arrested in the G2/M phase upon treatment with low doses of ISL. Mitochondrial membrane potential (DeltaPsim) disruption was observed at low ISL doses. The effect of ISL on DNA damage was analyzed by agarose gel electrophoresis and atomic force microscopy (AFM). Either ISL or Cu2+ failed to cause pBR322 DNA damage. However, plasmid DNA was damaged after treatment with ISL in the presence of Cu2+. Two forms of plasmid DNA closed circular and linear forms were visualized by AFM.

Studies on the cytotoxic activity of synthetic 2H-azirine-2-azetidinone compounds

2-Azetidinones and 2H-azirines show antibacterial and cytotoxic activities, however the biological properties of molecules containing both 2H-azirine and 2-azetidinone functions in the same structure had never been evaluated before. In the present study, two 2H-azirine-2-azetidinones ( 1 and 2) and three 2H-azirines ( 3– 5) were synthesized from 2-formyl-3-phenyl-2H-azirine-N-arylimines with diphenylketene. The compounds were assayed for antibacterial and cytotoxic activities. None of them showed antibacterial activity on the tested strains, but both 2H-azirine-2-azetidinones showed cytotoxicity against four tumor cell lines (HL-60, leukemia; HCT-8, colon cancer; MDA-MB-435, melanoma; and SF-295, CNS). The IC 50 values of 1 ranged from 1.1 to 10.5 μM and from 3.8 to 26.6 μM for 2. The mechanism of cell growth inhibition of 1 and 2 towards HL-60 cell line was also investigated. Membrane damage, cell viability, DNA synthesis inhibition and morphological changes were evaluated. The preliminary findings suggested that 1 and 2 induce apoptosis.

인간 남성호르몬 비의존형 전립선 PC-3 암세포에서 감마선의 Caspase-의존성 세포자멸사 유도 효과

Prostate cancer is the most predominant cancer in men and related death rate increases every year. Till date, there is no effective therapy for androgen independent prostate cancer. To investigate the mechanism for cell growth inhibition or apoptosis in human androgen independent prostate cancer PC-3 cells after gamma irradiation. The aim of this study was to examine the potential of gamma irradiation to induce apoptosis in PC-3 cells and to assess the mechanism of gamma irradiation-induced apoptosis. Five different assays were employed in this study: cell proliferation assay, morphological assessments of apoptotic cells, DNA fragmentation analysis, quantification of apoptosis by annexin V (AV) and propidium iodide (PI) staning, and western blot analysis. Cell viability was inversely related to radiation dose. DAPI-positive cells were detected 48 hr after 40 Gy radiation exposure. And nuclear morphological changes of cells were observed by gamma irradiation. DNA ladder patterns in the cells exposed to gamma-radiation were appeared at 24 hr. Also, gamma irradiation induces apoptosis of PC-3 cells via Caspase3, Bax and PARP-dependent fashion.

Covalent Binding to Tubulin by Isothiocyanates: A MECHANISM OF CELL GROWTH ARREST AND APOPTOSIS

Isothiocyanates (ITCs) found in cruciferous vegetables, including benzyl-ITC (BITC), phenethyl-ITC (PEITC), and sulforaphane (SFN), inhibit carcinogenesis in animal models and induce apoptosis and cell cycle arrest in various cell types. The biochemical mechanisms of cell growth inhibition by ITCs are not fully understood. Our recent study showed that ITC binding to intracellular proteins may be an important initiating event for the induction of apoptosis. However, the specific protein target(s) and molecular mechanisms were not identified. In this study, two-dimensional gel electrophoresis of human lung cancer A549 cells treated with radiolabeled PEITC and SFN revealed that tubulin may be a major in vivo binding target for ITC. We examined whether binding to tubulin by ITCs could lead to cell growth arrest. The proliferation of A549 cells was significantly reduced by ITCs, with relative activities of BITC > PEITC > SFN. All three ITCs also induced mitotic arrest and apoptosis with the same order of activity. We found that ITCs disrupted microtubule polymerization in vitro and in vivo with the same order of potency. Mass spectrometry demonstrated that cysteines in tubulin were covalently modified by ITCs. Ellman assay results indicated that the modification levels follow the same order, BITC > PEITC > SFN. Together, these results support the notion that tubulin is a target of ITCs and that ITC-tubulin interaction can lead to downstream growth inhibition. This is the first study directly linking tubulin-ITC adduct formation to cell growth inhibition.

Anti-proliferative and pro-apoptotic effect of CPT13, a novel camptothecin analog, on human colon cancer HCT8 cell line

10-(2-Pyrazolyl-ethoxy)-(20 S)-camptothecin (CPT13) is a novel semi-synthetic analogue of camptothecin, our previous report had shown that it possessed higher in vitro cytoxicity activity towards human colon cancer HCT8 cell line than topotecan. In this study, the anti-proliferative effect of CPT13 on HCT8 cell line in vitro was analyzed. In order to further explore the underlying mechanism of cell growth inhibition of CPT13 towards HCT8 cell line, the cell cycle distribution, apoptosis proportion, the nuclei morphological changes and caspase-8 and caspase-3 activities were measured. Additionally the changes of mitochondrial morphology and membrane potential (Δ Ψm) were analyzed by atomic force microscopy (AFM) and flow cytometry, respectively. The results showed that CPT13 inhibited HCT8 cell growth by causing cell cycle arrest at G 2/M transition and induced apoptosis, as evidenced by the typical apoptotic morphology such as condensation and fragmentation of nuclei and formation of apoptotic bodies. The changes of mitochondrial morphology, dose-dependently decrease in Δ Ψm and the enhancement of caspase-8 and caspase-3 activities were observed in different concentrations of drug treatment group. Our results suggest that CPT13 induces apoptosis by alternations of mitochondrial transmembrane depolarization, activation of caspase-8 and caspase-3. Therefore, CPT13 appears to be a potent drug against human colon cancer via induction of apoptosis and may be used as an alternative drug to therapy cancer.

A novel zinc-chelating small molecule exhibits cancer cell growth inhibition in vitro and in vivo by induction of tumor suppressors early growth response 1 (Egr-1) and Krüppel-Like Factor 4 (KLF4)

14084 Background: Lead compound LT-253 was selected from a group of 2-indolyl imidazol [4,5-d] phenanthroline derivatives with anticancer activity. It shows potent and selective anti-proliferative activity against several human cancer types in vitro, and in vivo in xenograft mouse models of human colon carcinoma (HT-29) and non-small cell lung carcinoma (H460). Methods: The mechanism of cell growth inhibition of LT-253 was investigated in HT-29 colon cancer cells using the XTT cell proliferation assay, flow cytometry and apoptosis assays. In vitro and in vivo zinc chelation was determined by competition assays using fluorescent and chromophoric chelators. Gene expression studies were performed by human genome microarray analysis and confirmed by quantitative real- time PCR. The transcription factor activity profile of LT-253-treated cells was determined by a multiplex transcription factor array and electrophoretic mobility shift assays. The functional role of specific genes was evaluated by siRNA gene knock-down. Results: LT-253 functions as chelator of zinc in vitro, and of intracellular labile zinc in HT-29 cells. Moreover, LT-253-mediated HT-29 cell growth inhibition was reversed by zinc supplementation. Gene expression profiling confirmed sustained changes in zinc-sensitive genes such as metallothionine and several zinc transporters, but not copper-sensitive or iron-sensitive genes. LT-253 induces cancer cell growth inhibition primarily through G1/S phase cell cycle arrest. Gene expression and transcription factor activities of both Egr-1 and KLF4 are induced within 4 hr post LT-253 treatment. Moreover, increased expression of both Egr-1 and KLF4 is observed in LT-253-sensitive cancer cell lines of various origins. Importantly, Egr-1 and KLF4 gene knock-down by siRNA reversed the LT-253-mediated cell growth inhibition of HT-29 cells. Conclusion: Selective chelation of intracellular labile zinc pool by LT-253 triggers immediate induction of stress-responsive tumor suppressor Egr-1 and sustained induction of zinc-responsive tumor suppressor KLF4, leading to G1/S phase cell cycle arrest and inhibition of tumor growth. No significant financial relationships to disclose.

Molecular targeting therapy with angiotensin II receptor blocker for prostatic cancer

Angiotensin II (Ang-II) plays a key role as a vasoconstrictor in controlling blood pressure and electrolyte/fluid homeostasis. Recently it has also been shown that this peptide is a cytokine, acting as a growth factor in cardiovascular and stromal cells. In addition, the physiological function of Ang-II seems to be similar in prostate cancer and stromal cells. It is widely assumed that Ang-II facilitates the growth of both cells, and its receptor blockers (ARBs) have the potential to inhibit the growth of various cancer cells and tumors through the Ang-II receptor type 1 (AT1 receptor). The mechanism of cell growth inhibition by ARBs has been considered to be that of suppression of the signal transduction systems activated by growth factors or cytokines in prostate cancer cells, and suppression of angiogenesis. This review highlights the possible use of ARBs as novel agents for prostatic diseases including prostate cancer and benign hypertrophy, and covers related literature.

Phosphorylation regulates Myc expression via prolonged activation of the mitogen‐activated protein kinase pathway

We previously showed that prolonged and strong ERK phosphorylation induced by Compound 5 (Cpd 5), a Cdc25A protein phosphatase inhibitor, was involved in its mechanism of cell growth inhibition. To study the relationship between ERK phosphorylation and cell growth inhibition, we used Cpd 5 as a tool to investigate ERK-regulated c-Myc expression in Hep3B hepatoma cells. We found that ERK phosphorylation caused by Cpd 5 induced c-Myc phosphorylation, but suppressed c-Myc expression at the mRNA and protein levels. Furthermore, Cpd 5 inhibited c-Myc transcriptional activity and DNA binding ability, and this inhibition was antagonized by ERK kinase (MEK) inhibitor U-0126, implying that the ERK pathway was involved in regulating c-Myc expression. Since the participation of c-Myc protein in transcription requires its dimerization with Max protein, we examined the Myc-Max association in Cpd 5-treated cells and found that Cpd 5 suppressed Myc-Max dimerization. Transfection of Hep3B cells with mutated ERK (T188A/Y190F), which has lost its dual-phosphorylation sites, attenuated the actions of Cpd 5 on Myc-Max association. To further demonstrate whether Myc phosphorylation by Cpd 5-induced ERK activation was able to directly regulate c-myc gene expression, a chromatin immunoprecipitation (ChIP) assay was used to examine the binding of phospho-Myc to the c-myc promoter region. We found that phospho-Myc induced by Cpd 5 had lost its ability to bind to the c-myc promoter, whereas MEK inhibitor U-0126 antagonized this inhibitory effect. These data suggest that an increase in c-Myc phosphorylation in response to prolonged ERK phosphorylation negatively auto-regulates c-Myc gene expression, leading to the suppression of its target gene expression and cell cycle block.