Flavonoid-mediated tumor growth suppression demonstrated by in vivo study

Many of flavonoids as well as bioflavonoids extracted from higher plants, which were earlier revealed to have tumor cell growth suppression activity in vitro, were examined for their effect on tumors in vivo. Balb/c mice were inoculated i.p. with syngeneic tumor cells, meth/a, and then provided with flavonoids dissolved in their drinking water during the course of their survival time. Many flavonoids were effective in prolonging the survival period. Furthermore, flavonoids that did not show suppressive activities in the in vitro experiments were effective in the in vivo assay. The data suggested that sugar bonded to the A ring, which suppresses tumor growth inhibition in vitro, plays an important role. Many aglycones that were effective in the in vitro assay on the tumor growth suppression were not effective in the in vivo assay. The reason for this seems to be that most aglycones are unstable and thus break down in vivo. No acute nor chronic toxicity of flavonoids was observed in the mice.     

Oral silibinin inhibits in vivo human bladder tumor xenograft growth involving down-regulation of survivin.

PURPOSE: Chemoprevention is an upcoming approach to control bladder cancer, which is one of the commonly diagnosed malignancies showing recurrence rate of 70% or even higher. Recently, we observed the in vitro efficacy of silibinin, a flavanolignan, in human bladder transitional cell papilloma RT4 cells. Here, we investigated the antitumor efficacy and associated mechanisms of silibinin in RT4 tumor xenograft. EXPERIMENTAL DESIGN: RT4 tumor xenograft was implanted s.c. in athymic nude mice, and then animals were oral gavaged with silibinin at 100 and 200 mg/kg doses, 5 days/week for 12 weeks. Tumor growth, body weight, and diet consumption were recorded, and tumors were analyzed for proliferation, apoptosis, and angiogenesis biomarkers and molecular alterations by immunohistochemistry, immunoblot analysis, and ELISA. p53 small interfering RNA was used in cell culture to examine the role of p53 in survivin expression. RESULTS: Silibinin feeding inhibited tumor xenograft growth without any gross signs of toxicity. Silibinin decreased tumor volume by 51% to 58% (P 相似文献   

In vivo and in vitro ovarian carcinoma growth inhibition by a phosphatidylinositol 3-kinase inhibitor (LY294002).

Phosphatidylinositol 3-kinase (PI3-K) induces mitogenesis, cell growth, and cell transformation. Amplification of the gene encoding the P110alpha subunit likely is an important event in ovarian cancer progression, and PI3-K inhibitors are possible therapeutic agents for this disease. We evaluated effects of LY294002, a potent inhibitor of PI3-K, on growth of ovarian carcinoma in vivo and in vitro, and on ascites formation in vivo. Athymic mice were inoculated i.p. with the ovarian cancer cell line OVCAR-3. Seven days after inoculation, mice were treated with or without LY294002 (100 mg/kg of body weight) for 3 weeks. Body weight and abdominal circumference were measured twice weekly. At the end of the experiment, mice were sacrificed, ascites volume was measured, and tumors were excised. Mean tumor burden in the LY294002-treated group was reduced by approximately 65% versus controls. Virtually no ascites developed in the treatment group; mean volume of ascites in controls was 3.3 +/- 0.38 ml. OVCAR-3 cells also were cultured in vitro without and with LY294002 (1, 5, and 10 microM) for 24 h. The number of cells in 1, 5, and 10 microM LY294002-treated wells was reduced by 27, 56, and 75%, respectively, versus controls. In vivo and in vitro morphological studies demonstrated that LY294002 induced marked nuclear pyknosis and diminished cytoplasmic volume in the tumor cells, confirmed as apoptosis. Thus, LY294002 significantly inhibits growth and ascites formation of ovarian carcinoma in vivo and markedly inhibits ovarian cancer cell proliferation in vitro, suggesting an important role of PI3-K inhibitors as a potentially useful treatment for women with ovarian carcinoma.     

Involvement of Bcl-X(L) deamidation in E1A-mediated cisplatin sensitization of ovarian cancer cells

The adenovirus E1A protein has been shown to be involved in the potentiation of apoptosis induced by chemotherapeutic agents, yet the molecular events of E1A-mediated apoptosis are not very clear. A recent report has suggested that deamidation of the Bcl-X(L) protein inhibits its antiapoptotic ability and leads to apoptosis induced by alkylating agents in Rb-deficient tumor cells. Since Rb is known to interact with E1A, which interrupts Rb's normal function, we examined Bcl-X(L) deamidation and cell death induced by cisplatin in E1A transfectants. We found that the E1A transfectants became sensitive to cisplatin-induced apoptosis compared to the parental cells, SKOV3.ip1. Our data show that cisplatin treatment induced the modification of Bcl-X(L) in the E1A transfectants in dosage and time-dependent manner. Furthermore, phosphatase treatment had no effect on the level of Bcl-X(L) modification, whereas alkaline lysis buffer appeared to induce the same modification of Bcl-X(L). Ectopic expression of the deamidated forms of Bcl- X(L) in SKOV3.ip1 cells revealed that the modification to the Bcl- X(L) protein molecules was deamidation. Expression of the E1A mutant (dl1108) which contains deletion at CR2 domain suppressed Bcl-X(L) deamidation and apoptosis induced by cisplatin. We also found that expression of the nondeamidated Bcl-X(L) protected E1A transfectants from apoptosis. These findings suggest that Bcl-X(L) deamidation contributes to E1A-mediated cisplatin sensitization in SKOV3.ip1 cells.     

Marked suppression of tumor growth by FTY720 in a rat liver tumor model: the significance of down-regulation of cell survival Akt pathway

We aim to investigate the anticancer effect of a novel immunomodulator FTY720 on a rat orthotopic liver tumor model. A buffalo rat orthotopic liver tumor model was established by injection of a buffalo hepatoma cell line MH7777 into the right portal vein. FTY720 was administered by intraperitoneal injection starting at 10 days after tumor cell injection at a dosage of 5 mg/kg/day. FTY720 markedly suppressed tumor growth and inhibited tumor progression by selective induction of apoptosis of tumor cells via down-regulation of phospho-Akt(ser473) and up-regulation of cleaved caspase-3, together with decrease of focal adhesion kinase. Moreover, the proliferation index of tumor cells was significantly reduced to 15.92+/-5.03% by FTY720 compared with that of 42.92+/-4.47% in the control group (p<0.001). In addition, we confirmed that FTY720 caused no effect on infiltrated lymphocyte in tumor tissue. We conclude that FTY720 is an effective anticancer agent for liver tumor in a rat model without affecting the immune system of the host.     

Geranylgeranylated RhoB mediates suppression of human tumor cell growth by farnesyltransferase inhibitors.

Farnesyltransferase inhibitors (FTIs) are in clinical trials, but their mechanism of action is not fully understood. We have shown that FTI treatment rapidly elevates the level of geranylgeranylated RhoB in cells and that this event is sufficient to inhibit cell cycle transit and reverse malignant transformation without affecting normal cells. However, because these observations were made in rodent fibroblast models in which transformation was driven by defined genetic alterations, it remained to be established whether RhoB-GG was relevant to the antineoplastic effects of FTIs in human epithelial tumor cells with diverse genetic backgrounds. In this study, we show that elevated levels of RhoB-GG are sufficient to block the proliferation of FTI-sensitive but not FTI-resistant human carcinoma cells. RhoB-GG induced the cell cycle kinase inhibitor p21(WAF1) in a p53-dependent manner, similar to FTI treatment, but this event was dispensable because RhoB-GG could still inhibit the growth of p53-null cells that lacked p21WAF1 activation. Consistent with actions beyond G1-phase arrest, certain cell lines exhibited accumulation in G2-M phase or an increased apoptotic index in response to RhoB-GG. We concluded that RhoB-GG suppressed human tumor cell proliferation by more than one mechanism and that it promoted apoptosis as well as inhibited cell cycle transit in malignant epithelial cells. These findings suggest how FTIs suppress the growth of human tumor cells that lack Ras mutations.     

An in vivo mouse reporter gene (human secreted alkaline phosphatase) model to monitor ovarian tumor growth and response to therapeutics

PURPOSE: Developing new anticancer therapeutic regimens requires the measurement of tumor cell growth in response to treatment. This is often accomplished by injecting immunocompromised mice with cells from cancer tissue or cell lines. After treating the animals, tumor weight or volume is measured. Such methods are complicated by inaccuracies in measuring tumor mass and often animals must be killed to measure tumor burden. An in vivo tumor model system is presented in which the tumor cell line was stably transfected with a constitutively expressed marker gene: secreted human placental alkaline phosphatase protein (SEAP). The SEAP gene codes for a heat-stable protein that is produced at levels proportional to the amount of tumor cells in the animal. The SEAP protein is detectable in small blood samples so that animals can be repeatedly sampled over the trial period to monitor the course of tumor progression. METHODS: OCC1 ovarian carcinoma cells were stably transfected with pCMV-SEAP. The OCC1-SEAP cells were maintained in vitro to monitor the relationship between cell number and SEAP production. Experiments were performed in vivo to determine whether SEAP levels in blood corresponded to tumor burden. OCC1-SEAP cells were injected s.c. or intraperitoneally into nude mice and tumor volume was measured as well as plasma SEAP levels as the tumors developed. RESULTS: S.c. tumor volume correlated well with plasma SEAP levels ( R(2)=0.95). OCC1-SEAP cells were also injected intraperitoneally into nude mice and grown as abdominal tumors. After 3 weeks the animals were killed and the tumors were dissected and weighed. SEAP levels in plasma samples from the time of death correlated with intraperitoneal tumor weight ( R(2)=0.87). Experiments were performed to determine whether measuring SEAP levels could be used to monitor ovarian carcinoma cell response to platinum-containing chemotherapeutic drugs. OCC1-SEAP cells cultured in vitro were treated with the platinum-containing drug carboplatin. Carboplatin treatment decreased both cell proliferation and SEAP levels in culture medium. The constitutive rate of SEAP secretion per cell (nanograms SEAP per microgram DNA) was found not to be altered by carboplatin treatment. Therefore changes in SEAP level reflect changes in OCC1 tumor cell number, and not changes in regulation of SEAP secretion due to platinum containing chemotherapeutic drug treatment. OCC1 cells were injected intraperitoneally into nude mice and the mice were treated with the platinum-containing drugs cisplatin or carboplatin. Measurements of plasma SEAP over the treatment period showed OCC1-SEAP ovarian carcinoma growth to be inhibited by cisplatin and carboplatin treatment. CONCLUSION: The SEAP marker protein is constitutively expressed by tumor cells and blood levels are correlated with tumor cell number and burden. The results of these studies indicate that SEAP may be used as an in vivo reporter gene in a mouse model to monitor tumor growth and response to therapeutics. Future studies will utilize this model to investigate novel chemotherapeutic approaches to treating ovarian cancer.     

Blocking CXCR4-mediated cyclic AMP suppression inhibits brain tumor growth in vivo

The chemokine CXCL12 and its cognate receptor CXCR4 regulate malignant brain tumor growth and are potential chemotherapeutic targets. However, the molecular basis for CXCL12-induced tumor growth remains unclear, and the optimal approach to inhibiting CXCR4 function in cancer is unknown. To develop such a therapeutic approach, we investigated the signaling pathways critical for CXCL12 function in normal and malignant cells. We discovered that CXCL12-dependent tumor growth is dependent upon sustained inhibition of cyclic AMP (cAMP) production, and that the antitumor activity of the specific CXCR4 antagonist AMD 3465 is associated with blocking cAMP suppression. Consistent with these findings, we show that pharmacologic elevation of cAMP with the phosphodiesterase inhibitor Rolipram suppresses tumor cell growth in vitro and, upon oral administration, inhibits intracranial growth in xenograft models of malignant brain tumors with comparable efficacy to AMD 3465. These data indicate that the clinical evaluation of phosphodiesterase inhibitors in the treatment of patients with brain tumors is warranted.     

Effect of allomelanin on tumor growth suppression in vivo and on the cell cycle phase

Allomelanins are nitrogen-free macromolecular polymers of simple phenols produced by higher plants and fungi. In an earlier work we found that allomelanins were effective in suppressing the growth of cultured tumor cells. In the present study we examined the effect of these polymers on the survival curve of Balb/C mice inoculated i.p. with Meth/A cells (3 x 10(4) cells/mouse), which originated from a malignant lymphoma. Allomelanins were extracted from black sesame seeds and black soybeans, and given p.o. via the drinking water. The dose of allomelanins was approximately 3 mg/mouse/day. The percent of survivals was significantly higher in the experimental groups than in the control. Also, when HCT-15 cells were cultured for 2 days in medium containing 400 micrograms/ml of protein-free allomelanins, histograms obtained from flow-cytometry data showed a significant increase in the fraction between the diploid and tetraploid peaks, indicating blockage of the S phase of the cell cycle. These results suggest that allomelanins suppressed the tumor cell growth in vitro and in vivo, and that the effect was cytostatic, mostly by blockage of the S phase.     

Inhibition of p38alpha MAPK enhances proteasome inhibitor-induced apoptosis of myeloma cells by modulating Hsp27, Bcl-X(L), Mcl-1 and p53 levels in vitro and inhibits tumor growth in vivo.

Inhibition of p38 kinase blocks the production of tumor-promoting factors in the multiple myeloma (MM) bone marrow microenvironment. Proteasome inhibitors MG132 and bortezomib have been shown to have direct cytotoxic effects on MM cells. We show that a selective inhibitor of p38alpha, SCIO-469, enhances the ability of MG132 and bortezomib to induce the apoptosis of MM cells. Previously, we showed that p38 inhibition with SCIO-469 enhances MM cytotoxicity of bortezomib by inhibiting the transient expression and phosphorylation of Hsp27, a downstream target of p38. Here we show that continued treatment of MM cells with bortezomib leads to a SCIO-469-enhanced downregulation of Hsp27 and to increased MM apoptosis. Furthermore, we show that p38 inhibition enhances the bortezomib-induced MM apoptosis by upregulation of p53 and downregulation of Bcl-X(L) and Mcl-1. In a mouse xenograft plasmacytoma model of MM, we found that inhibiting p38 augments the effects of bortezomib in decreasing MM tumor growth in vivo. Thus, in addition to its role in suppressing an activated MM microenvironment, co-treatment with a p38 inhibitor, such as SCIO-469, may enhance the cytotoxicity of bortezomib by modulating pro-apoptotic and anti-apoptotic factors in MM cells, suggesting great potential for co-therapy.     

Stable RNA interference-mediated suppression of cyclophilin A diminishes non-small-cell lung tumor growth in vivo

Cyclophilin A (CypA) was recently reported to be overexpressed in non-small-cell lung cancer, and represents a potentially novel therapeutic target. To determine the role of CypA in oncogenesis, stable RNA interference (RNAi)-mediated knockdown of CypA was established in two non-small-cell lung cancer cell lines (ADLC-5M2 and LC-103H), and these cells were grown as xenografts in severe combined immunodeficient mice. Tumor cell proliferation, apoptosis, and angiogenesis were measured by Ki67, terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling, and CD31 immunohistochemistry, respectively. Tumor glucose metabolism was assessed by fluorodeoxyglucose positron emission tomography imaging. Knockdown of CypA correlated in vivo with slower growth, less fluorodeoxyglucose uptake, decreased proliferation, and a greater degree of apoptosis in the tumors. These results establish the relevance of CypA to tumor growth in vivo, specifically to proliferation and apoptosis. Elucidation of the precise role of CypA in these pathways may lead to new targeted therapies for lung cancer.     

Circulating mucins as tumor markers in ovarian cancer (review).

Because a highly sensitive and specific serum marker for ovarian carcinoma has not been reported, it is unlikely that there will be an application of serum markers for screening for this disease in asymptomatic women. However, many oncologists use serum tumor markers initially to differentiate epithelial ovarian carcinoma from benign gynecological conditions prior to surgery, so as to ensure appropriate surgical referral, and then to monitor the clinical course of disease during and after adjuvant therapy. The most commonly performed tumor marker assay in ovarian cancer (CA125) has been extremely valuable in patient management, but this marker is also elevated in a considerable proportion of patients with benign gynecologic diseases and endometriosis, and a relatively small proportion of patients with early stage disease. A new class of serum tumor markers, the highly glycosylated, high molecular weight mucins, have enormous potential in the management of ovarian cancer patients, since the use of assays for these markers may overcome many of the problems associated with CA125. Indeed, when used in combination with CA125, some mucin-based assays have increased the sensitivity and specificity of detection, thereby eliminating many false positive results seen with patients with benign disease and endometriosis, and also predicted disease recurrence in the majority of patients before clinical symptoms became apparent. These markers are the subject of this review, with particular attention to the commercially-available mucin-based assays.     

Bcl-X(L) is a chemoresistance factor in human melanoma cells that can be inhibited by antisense therapy

Malignant melanoma is a tumor that responds poorly to a variety of apoptosis-inducing treatment modalities, such as chemotherapy. The expression of genes that regulate apoptotic cell death plays an important role in determining the sensitivity of tumor cells to chemotherapeutic intervention. Bcl-x(L) is an antiapoptotic member of the Bcl-2 family and is universally expressed in human melanoma. To evaluate the Bcl-x(L) protein as a potential therapeutic target in melanoma, the influence of Bcl-x(L) expression levels on the chemoresistance of human melanoma cells was investigated. Overexpression of Bcl-x(L) in stably transfected human melanoma Mel Juso cells significantly reduced sensitivity to cisplatin-induced apoptosis (p < or = 0.05). In a parallel approach, reduction of Bcl-x(L) protein by specific AS oligonucleotide (ISIS 16009) treatment enhanced the chemosensitivity of Mel Juso cells by 62% compared to cells treated with MM control oligonucleotide (ISIS 16967) as well as chemotherapy-induced apoptosis. These data suggest that Bcl-x(L) is an important factor contributing to the chemoresistance of human melanoma. Reduction of Bcl-x(L) expression by AS oligonucleotides provides a rational and promising approach that may help to overcome chemoresistance in this malignancy.     

Cell cycle genes as targets of retinoid induced ovarian tumor cell growth suppression.

We have examined the effect of all-trans-retinoic acid (RA) on cell cycle gene expression in RA sensitive CA-OV3 and RA resistant SK-OV3 ovarian carcinoma cell lines. Gene expression was analysed by multiprobe RNAse protection, Western blotting and in vitro kinase assays. No differences were observed between RA sensitive and RA resistant ovarian carcinoma cells in the levels of expression of many cell cycle genes including cyclin A, B and E, cdk 2,4 and 6, E2F-1, E2F-2, E2F-3, E2F-4, E2F-5, DP-1 and DP-2. However, RA sensitive CA-OV3 cells expressed higher levels of p53, p27, p21, and p16 compared to RA resistant SK-OV3 cells. In addition, RA treatment of CA-OV3 cells resulted in a significant decrease in hyperphosphorylated RB and RB-2/p130 and corresponding significant increases in the levels of hypophosphorylated and/or partially phosphorylated RB-2/p130 protein and hypophosphorylated RB. Also, RA treatment increased expression of the cdk inhibitor p27 and decreased activity of cdk 2, cdk 4 and cdk 6. Finally, amounts of p27-cyclin E and RB-2/p130-E2F4 complexes were found to increase in CA-OV3 cells growth arrested by RA. These results suggest that the pocket protein pathways are critical targets for retinoid suppression of ovarian carcinoma cell growth.     

Genes associated with tumor suppression and growth control in the human nervous system

Cancer, the uncontrolled proliferation of a population of somatic cells, is fundamentally a genetic disorder. Although the specific array of genetic changes causing individual tumor types remains largely obscure, the past two decades have witnessed a tremendous increase in our understanding of the specific genes regulating cell differentiation, proliferation, and senescence. There appear to be two distinct fundamental genetic mechanisms of tumorigenesis. One mechanism is associated with the activation of growth-promoting factors such as proto-oncogenes. Altermatively, tumor formation may be induced as the result of the loss or inactivation of genes which normally regulate or suppress cell growth. These genes have been termed tumor suppressor genes or anti-oncogenes. This review focuses on the role of tumor suppressor genes in tumor formation and growth control of the human nervous system.     

Characterization of in vivo suppression of syngenic tumor by allogenic effector cells

The purpose of this study was to characterize the effects of allogenic splenocytes transferred with tumor cells, subcutaneously, to a host syngenic for tumor cells. Cytotoxic effector cells usually resulted in tumors less than half the size of controls at E:T of 10:1. Primary immune splenocytes were more effective than hyperimmune splenocytes in delaying tumor growth. Actively cytotoxic splenocytes by in vitro ⁵¹Cr release assay were required for delayed tumor growth; memory cell populations were not effective. Delayed tumor growth correlated with in vitro cytotoxicity of primary immune splenocytes; however, hyperimmune splenocytes, even though they possessed greater in vitro cytotoxic responses, showed lesser tumor suppression in vivo. T cells were necessary for tumor suppresion, as treatment of B6AF1 effector cells with anti-Thy 1.2 serum abrogated suppression; T cell enrichement by nylon-wool treatment of effector cells increased tumor suppression. Delay in tumor growth was an in vivo phenomenon, for anti-Thy 1.2 serum in AKR hosts abrogated the effect of Thy 1.2 effector cells.