Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells

Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 10⁹ CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 10⁹ CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain.     

In vitro induction of primary and secondary xenoimmune responses by liposomes containing human colon tumor cell antigens

Liposomes prepared with human LS174T colon tumor cell membranes induce specific primary and secondary xenogeneic immune responses in BALB/c splenocytes in vitro. The multilamellar vesicular liposomes were prepared by adding sonicated membrane fragments in 8 mM CaCl₂ to a dried lipid film. Cytotoxic splenocytes generated in vivo exhibited specificity for the LS174T cell; liposomes elicited higher levels of cytotoxicity than did membranes (P < 0.01). Secondary blastogenic responses elicited in in vivo-primed spleen cells by liposome-antigens also produced a significantly greater (P < 0.005) response than membranes. Subsequently, in vitro induction of primary blastogenic and cytotoxic responses by liposome-antigens were accomplished and revealed similar kinetics to that of whole LS174T cell immunogens. Specificity of the in vitro-primed spleen cells was clearly demonstrated (P < 0.01) on a variety of human tumor cells using both the primed lymphocyte and cell-mediated cytotoxicity assays. The results of competitive inhibition tests with autologous lymphoblasts demonstrated that 30% of the cytotoxic activity was directed against lymphocyte antigens. Incorporation of tumor antigens into liposomes has thus enabled primary immunization in vitro to human colon cancer antigens and may afford an adaptable means to evaluate and to select desired immune responses, as well as to identify colon tumor-specific determinants.     

The research on the mechanism of Tsoong inhibiting for colon cancer

The roots of Codonopis bulleynana Forest ex diels (cbFed), locally known as Tsoong, have been used as a tonic food. Tsoong has wide range of pharmacological effects, including anticancer effects. In the present study, the anticancer activity of Tsoong and its potential molecular mechanisms were investigated. Using high throughput sequencing the apoptotic pathway was ranked as one of the most important pathways and the differential expressions of apoptosis-related genes such as Casp3, Casp6 and Apaf1 were identified. The following experiments were qRT-PCR which were used to verify the genes. In vitro, cell counting kit-8 (CCK-8) assays and flow cytometry in HCT116 and SW480 colon cancer cell were used to assess the anti‐proliferation and apoptosis-promoting activities of Tsoong. In vivo, the antitumor effect of Tsoong was assessed in colon cancer-bearing nude mice as a xenograft model. H&E staining was performed with oxaliplatin set as a positive control. The results showed that Tsoong up-regulated apoptosis-related genes, inhibited tumor cell proliferation, promoted tumor cellapoptosis in a dose-dependent manner and restrained the growth of colon neoplasm. The effects of a high dose of Tsoong on colon cancer cells were similar to those of oxaliplatin. Our results may ultimately help in the development of diagnostic and therapeutic strategies to control this devastating disease. Therefore, Tsoong may be a promising Chinese herbal compound for development for use in cancer therapy.     

Differential Antitumor Effects of IgG and IgM Monoclonal Antibodies and Their Synthetic Complementarity-Determining Regions Directed to New Targets of B16F10-Nex2 Melanoma Cells

Malignant melanoma has increased incidence worldwide and causes most skin cancer-related deaths. A few cell surface antigens that can be targets of antitumor immunotherapy have been characterized in melanoma. This is an expanding field because of the ineffectiveness of conventional cancer therapy for the metastatic form of melanoma. In the present work, antimelanoma monoclonal antibodies (mAbs) were raised against B16F10 cells (subclone Nex4, grown in murine serum), with novel specificities and antitumor effects in vitro and in vivo. MAb A4 (IgG2ak) recognizes a surface antigen on B16F10-Nex2 cells identified as protocadherin β₁₃. It is cytotoxic in vitro and in vivo to B16F10-Nex2 cells as well as in vitro to human melanoma cell lines. MAb A4M (IgM) strongly reacted with nuclei of permeabilized murine tumor cells, recognizing histone 1. Although it is not cytotoxic in vitro, similarly with mAb A4, mAb A4M significantly reduced the number of lung nodules in mice challenged intravenously with B16F10-Nex2 cells. The V_H CDR3 peptide from mAb A4 and V_L CDR1 and CDR2 from mAb A4M showed significant cytotoxic activities in vitro, leading tumor cells to apoptosis. A cyclic peptide representing A4 CDR H3 competed with mAb A4 for binding to melanoma cells. MAb A4M CDRs L1 and L2 in addition to the antitumor effect also inhibited angiogenesis of human umbilical vein endothelial cells in vitro. As shown in the present work, mAbs A4 and A4M and selected CDR peptides are strong candidates to be developed as drugs for antitumor therapy for invasive melanoma.     

Cancer-initiating cells derived from human rectal adenocarcinoma tissues carry mesenchymal phenotypes and resist drug therapies

Accumulating evidence indicates that cancer-initiating cells (CICs) are responsible for cancer initiation, relapse, and metastasis. Colorectal carcinoma (CRC) is typically classified into proximal colon, distal colon, and rectal cancer. The gradual changes in CRC molecular features within the bowel may have considerable implications in colon and rectal CICs. Unfortunately, limited information is available on CICs derived from rectal cancer, although colon CICs have been described. Here we identified rectal CICs (R-CICs) that possess differentiation potential in tumors derived from patients with rectal adenocarcinoma. The R-CICs carried both CD44 and CD54 surface markers, while R-CICs and their immediate progenies carried potential epithelial–mesenchymal transition characteristics. These R-CICs generated tumors similar to their tumor of origin when injected into immunodeficient mice, differentiated into rectal epithelial cells in vitro, and were capable of self-renewal both in vitro and in vivo. More importantly, subpopulations of R-CICs resisted both 5-fluorouracil/calcium folinate/oxaliplatin (FolFox) and cetuximab treatment, which are the most common therapeutic regimens used for patients with advanced or metastatic rectal cancer. Thus, the identification, expansion, and properties of R-CICs provide an ideal cellular model to further investigate tumor progression and determine therapeutic resistance in these patients.     

Treatment with Insulin Analog X10 and IGF-1 Increases Growth of Colon Cancer Allografts

Obesity and type 2 diabetes are associated with an increased risk for development of certain forms of cancer, including colon cancer. The publication of highly controversial epidemiological studies in 2009 raised the possibility that use of the insulin analog glargine increases this risk further. However, it is not clear how mitogenic effects of insulin and insulin analogs measured in vitro correlate with tumor growth-promoting effects in vivo. The aim of this study was to examine possible growth-promoting effects of native human insulin, insulin X10 and IGF-1, which are considered positive controls in vitro, in a short-term animal model of an obesity- and diabetes-relevant cancer. We characterized insulin and IGF-1 receptor expression and the response to treatment with insulin, X10 and IGF-1 in the murine colon cancer cell line (MC38 cells) in vitro and in vivo. Furthermore, we examined pharmacokinetics and pharmacodynamics and monitored growth of MC38 cell allografts in mice with diet-induced obesity treated with human insulin, X10 and IGF-1. Treatment with X10 and IGF-1 significantly increased growth of MC38 cell allografts in mice with diet-induced obesity and we can therefore conclude that supra-pharmacological doses of the insulin analog X10, which is super-mitogenic in vitro and increased the incidence of mammary tumors in female rats in a 12-month toxicity study, also increase growth of tumor allografts in a short-term animal model.     

Cytotoxic Neo‐Clerodane Diterpenoids from Scutellaria barbata D.Don

A new neo‐clerodane diterpenoid, barbatin H ( 1 ), together with fifteen known analogues ( 2 – 16 ) were isolated from Scutellaria barbata D.Don . Their structures were determined on the basis of NMR and HR‐MS spectral analysis and comparison with the reported data. All of those compounds were comparatively predicted for their cytotoxic activities against four human tumor cell lines, i. e. LoVo (colon cancer), MCF‐7 (breast cancer), SMMC‐7721 (hepatoma cancer), and HCT‐116 (colon cancer) cells by MTT method in vitro. The results turned out that the series of neo‐clerodane diterpenoids exhibited varying degrees of cytotoxic activities against the growth of the tested tumor cell lines, and most of them exhibited selective cytotoxicity against LoVo cell lines. Scutebata A ( 14 ) showed significant cytotoxic activities against four tested tumor cells with IC₅₀ values of 4.57, 7.68, 5.31, and 6.23 μm , respectively, which indicated that it might be a potential chemotherapeutic agent.     

Analysis of Quinolinequinone Analogs with Promising Cytotoxic Activity against Breast Cancer

It is quite challenging to find out bioactive molecules in the vast chemical universe. Quinone moiety is a unique structure with a variety of biological properties, particularly in the treatment of cancer. In an effort to develop potent and secure antiproliferative lead compounds, five quinolinequinones ( AQQ1-5 ) described previously have been selected and submitted to the National Cancer Institute (NCI) of Bethesda to envisage their antiproliferative profile based on the NCI Developmental Therapeutics Program. According to the preliminary in vitro single-dose anticancer screening, four of five quinolinequinones ( AQQ2-5 ) were selected for five-dose screening and they displayed promising antiproliferative effects against several cancer types. All AQQs showed a excellent anticancer profile with low micromolar GI₅₀ and TGI values against all leukemia cell lines, some non-small cell lung and ovarian cancer, most colon, melanoma, and renal cancer, and in addition to some breast cancer cell lines. AQQ2-5 reduced the proliferation of all leukemia cell lines at a single dose and five additional doses, as well as some non-small cell lung and ovarian cancer, the majority of colon cancer, melanoma and renal cancer, and some breast cancer cell lines. This motivated us to use in vitro, in silico, and in vivo technologies to further investigate their mode of action. We investigated the in vitro cytotoxic activities of the most promising compounds, AQQ2 and AQQ3 , in HCT-116 colon cancer, MCF7 and T-47D breast cancer, and DU-145 prostate cancer cell lines, and HaCaT human keratinocytes. Concomitantly, IC₅₀ values of AQQ2 and AAQ3 against MCF7 and T-47D cell lines of breast cancer, DU-145 cell lines of prostate cancer, HCT-116 cell lines of colon cancer, and HaCaT human keratinocytes were determined. AQQ2 exhibited anticancer activity through the induction of apoptosis and caused alterations in the cell cycle. In silico pharmacokinetic studies of all analogs have been carried out against ATR, CHK1, WEE1, CDK1, and CDK2. In addition to this, in vitro ADME and in vivo pharmacokinetic profiling for the most effective AAQ ( AAQ2 ) have been studied.     

Subpopulations of human T lymphocytes. II. Effect of thymopoietin, corticosteroids, and irradiation.

Lymphoid cells from normal SJL/J mice gave high proliferative responses but failed to develop cytotoxic activity to γ-irradiated cells from syngeneic transplantable reticulum cell sarcomas (X-RCS). In spite of a vigorous in vivo proliferative response to X-RCS, cytotoxic activity was never generated to detectable levels in vivo. After repeated injections of X-RCS, spleen and, to a lesser degree, lymph node cells acquired the ability to give moderate secondary cytotoxic responses in vitro upon co-culture with X-RCS. This immunity was T-cell mediated and specific for RCS although it did not distinguish between different transplantable RCS lines. SJL/J mice also developed resistance to RCS growth after injection of X-RCS, which showed a transient RCS-line-specific component. (SJL/J × C₅₇B1/6)F₁ mice showed 60% less RCS growth than did SJL/J mice, and their lymphoid cells gave slightly lower proliferative responses than did cells from SJL/J mice, whereas (SJL/J × BALB/c)F₁ mice showed little tumor growth, and their spleen cells proliferated only minimally to X-RCS. B10.S mice allowed moderate RCS growth. Cytotoxic activity was generated in co-cultures with X-RCS of immunized F₁ spleen cells even after a single immunization in vivo but not in cultures of normal F₁ cells with X-RCS.     

Omega-3 Eicosapentaenoic Acid Decreases CD133 Colon Cancer Stem-Like Cell Marker Expression While Increasing Sensitivity to Chemotherapy

Colorectal cancer is the third leading cause of cancer-related death in the western world. In vitro and in vivo experiments showed that omega-3 polyunsaturated fatty acids (n-3 PUFAs) can attenuate the proliferation of cancer cells, including colon cancer, and increase the efficacy of various anticancer drugs. However, these studies address the effects of n-3 PUFAs on the bulk of the tumor cells and not on the undifferentiated colon cancer stem-like cells (CSLCs) that are responsible for tumor formation and maintenance. CSLCs have also been linked to the acquisition of chemotherapy resistance and to tumor relapse. Colon CSLCs have been immunophenotyped using several antibodies against cellular markers including CD133, CD44, EpCAM, and ALDH. Anti-CD133 has been used to isolate a population of colon cancer cells that retains stem cells properties (CSLCs) from both established cell lines and primary cell cultures. We demonstrated that the n-3 PUFA, eicosapentaenoic acid (EPA), was actively incorporated into the membrane lipids of COLO 320 DM cells. 25 uM EPA decreased the cell number of the overall population of cancer cells, but not of the CD133 (+) CSLCs. Also, we observed that EPA induced down-regulation of CD133 expression and up-regulation of colonic epithelium differentiation markers, Cytokeratin 20 (CK20) and Mucin 2 (MUC2). Finally, we demonstrated that EPA increased the sensitivity of COLO 320 DM cells (total population) to both standard-of-care chemotherapies (5-Fluorouracil and oxaliplatin), whereas EPA increased the sensitivity of the CD133 (+) CSLCs to only 5-Fluorouracil.     

Src-Mediated Cross-Talk between Farnesoid X and Epidermal Growth Factor Receptors Inhibits Human Intestinal Cell Proliferation and Tumorigenesis

Besides its essential role in controlling bile acid and lipid metabolism, the farnesoid X receptor (FXR) protects against intestinal tumorigenesis by promoting apoptosis and inhibiting cell proliferation. However, the mechanisms underlying these anti-proliferative actions of FXR remain to be elucidated. In the present study, we examined the effects of FXR activation (FXR overexpression and treatment with an FXR agonist GW4064) and inactivation (treatment with FXR siRNA and an FXR antagonist guggulsterone) on colon cancer cell proliferation in vitro using human colon cancer cell lines (H508, SNU-C4 and HT-29) and in vivo using xenografts in nude mice. Blocking FXR activity with guggulsterone stimulated time- and dose-dependent EGFR (Tyr845) phosphorylation and ERK activation. In contrast, FXR overexpression and activation with GW4064 attenuated cell proliferation by down-regulating EGFR (Tyr845) phosphorylation and ERK activation. Treatment with guggulsterone and GW4064 also caused dose-dependent changes in Src (Tyr416) phosphorylation. In stably-transfected human colon cancer cells, overexpression of FXR reduced EGFR, ERK, Src phosphorylation and cell proliferation, and in nude mice attenuated the growth of human colon cancer xenografts (64% reduction in tumor volume; 47% reduction in tumor weight; both P<0.01). Moreover, guggulsterone-induced EGFR and ERK phosphorylation and cell proliferation were abolished by inhibiting activation of Src, EGFR and MEK. Collectively these data support the novel conclusion that in human colon cancer cells Src-mediated cross-talk between FXR and EGFR modulates ERK phosphorylation, thereby regulating intestinal cell proliferation and tumorigenesis.     

Protein Expression Profile of HT-29 Human Colon Cancer Cells after Treatment with a Cytotoxic Daunorubicin-GnRH-III Derivative Bioconjugate

Targeted delivery of chemotherapeutic agents is a new approach for the treatment of cancer, which provides increased selectivity and decreased systemic toxicity. We have recently developed a promising drug delivery system, in which the anticancer drug daunorubicin (Dau) was attached via oxime bond to a gonadotropin-releasing hormone-III (GnRH-III) derivative used as a targeting moiety (Glp-His-Trp-Lys(Ac)-His-Asp-Trp-Lys(Dau = Aoa)-Pro-Gly-NH₂; Glp = pyroglutamic acid, Ac = acetyl; Aoa = aminooxyacetyl). This bioconjugate exerted in vitro cytostatic/cytotoxic effect on human breast, prostate and colon cancer cells, as well as significant in vivo tumor growth inhibitory effect on colon carcinoma bearing mice. In our previous studies, H-Lys(Dau = Aoa)-OH was identified as the smallest metabolite produced in the presence of rat liver lysosomal homogenate, which was able to bind to DNA in vitro. To get a deeper insight into the mechanism of action of the bioconjugate, changes in the protein expression profile of HT-29 human colon cancer cells after treatment with the bioconjugate or free daunorubicin were investigated by mass spectrometry-based proteomics. Our results indicate that several metabolism-related proteins, molecular chaperons and proteins involved in signaling are differently expressed after targeted chemotherapeutic treatment, leading to the conclusion that the bioconjugate exerts its cytotoxic action by interfering with multiple intracellular processes.     

Knockdown of Lymphoid Enhancer factor 1 Inhibits Colon Cancer Progression In Vitro and In Vivo

Expression of lymphoid enhancer factor 1 (LEF1) is frequently altered in different human cancers. This study aimed to assess LEF1 expression in colon cancer tissues and to explore changed phenotypes, gene expressions, and the possible mechanism after knocked down LEF1 expression in colon cancer cell lines. A total of 106 colon cancer and matched paratumorous normal tissues were used to assess LEF1 expression using immunohistochemistry and qRT-PCR. LEF1 lentivirus was used to knockdown LEF1 expression for the assessment of cell viability, cell cycle distribution, apoptosis, and gene expressions. The nude mouse xenograft assay was performed to detect the effects of LEF1 knockdown in vivo. The data showed that the levels of LEF1 mRNA and protein were significantly increased in human colon cancer tissues compared to the matched paratumorous normal tissues and were associated with infiltration depth, lymph node and distant metastases, advanced TNM (tumor-node-metastasis) stages, and shorter overall survival. Furthermore, LEF1 knockdown reduced tumor cell viability, invasion capacity, MMP2 and MMP-9 expression, but induced apoptosis. Nude mouse xenograft assay showed that LEF1 knockdown suppressed tumor formation and growth in vivo. In addition, the expression of Notch pathway-related proteins RBP-jκ and Hes1 was reduced in LEF1 knockdown cells. Taken together, LEF1 protein was overexpressed in colon cancer tissues and knockdown of LEF1 expression inhibited colon cancer growth in vitro and in vivo. These data suggest that targeting of LEF1 expression should be further evaluated for colon cancer prevention and therapy.     

Evaluations of Anticancer Effects of Combinations of Cisplatin and Tirucallane-Type Triterpenes Isolated from Amphipterygium adstringens (Schltdl).

The cytotoxic activity of combinations of masticadienonic (AMD) or 3αOH-hydroxy-masticadienonic (3αOH-AMD) acids with cisplatin (CDDP) was evaluated against PC3 prostate and HCT116 colon cancer cell lines. Combinations A (half the IC₅₀ value), B (IC₅₀ value), and C (twice the IC₅₀ value) were tested at a 1 : 1 ratio. All AMD plus CDDP combinations demonstrated increased cytotoxic effect, as determined by the sulforhodamine B test, in both cell types. The best combination was B, which showed 93 % and 91 % inhibition of the proliferation of PC3 and HCT116 cells, respectively. It also increased apoptosis in the PC3 cell lines, as evaluated by flow cytometry. However, in vivo tests showed no additional activity from the AMD plus CDDP combinations. These results showed that the increased cytotoxic activity of the combinations in vitro did not reflect in vivo tests. All combinations of 3αOH-AMD plus CDDP exerted antagonistic effects in both cell types.     

Studies on the specificity of in vitro-induced lymphocytotoxicity to methylcholanthrene-induced fibrosarcoma cell lines

Cytotoxic effector lymphocytes were induced by in vitro immunization of lymph node and spleen cells from CS7B16(H2^b) and Balb/c(H2^d) mice to syngeneic or allogeneic methylcholanthrene-induced fibrosarcoma (MCAF) cell lines. The T cell-dependent cytotoxicity was specific to target cell lines to which the lymphocytes were immunized in vitro. Normal fibroblasts as stimulator cells did not induce lymphocytotoxicity to syngeneic MCAF cells or to normal syngeneic fibroblasts. The results indicate that the in vitro-immunized lymphocytes recognize individual specific tumor-associated antigens of the MCAF cells. In experiments in which the lymphocytes were immunized in vitro to allogeneic MCAF cells, cytotoxic reactions to alloantigens, but not to tumor-associated antigens, were detected. Incubation with phytohemagglutinin (PHA) during the sensitization period modified the specificity of the cell-mediated lysis of MCAF cells: Allogeneic as well as syngeneic target cells were destroyed by these effector cells. PHA induced a nonspecific cytotoxic effect which increased the specific lysis of target cells. The cytotoxicity of the in vitro-immunized lymphocytes was inhibited by incubation with membrane protein preparations from the syngeneic MCAF cell lines. In contrast to the specificity of the cytotoxic effect to the different syngeneic cell lines, the membrane extract of one individual syngeneic MCAF cell line was able to inhibit the lymphocytotoxicity to all other syngeneic cell lines. Membrane protein preparations from allogeneic MCAF cells or from normal syngeneic fibroblasts were not inhibitory. The in vitro-immunized cytotoxic lymphocytes did not impair the tumor growth in vivo as could be demonstrated by passive transfer of the lymphocytes in a Winn assay.     

Egr-1 is required for neu/HER2-induced mammary tumors

Egr-1 is known to function mainly as a tumor suppressor through direct regulation of multiple tumor suppressor genes. To determine the role of Egr-1 in breast tumors in vivo, we used mouse models of breast cancer induced by HER2/neu. We compared neu-overexpressing Egr-1 knockout mice (neu/Egr-1 KO) to neu-overexpressing Egr-1 wild type or heterozygote mice (neu/Egr-1 WT or neu/Egr-1 het) with regard to onset of tumor appearance and number of tumors per mouse. In addition, to examine the role of Egr-1 in vitro, we established neu/Egr-1 WT and KO tumor cell lines derived from breast tumors developed in each mouse. Egr-1 deletion delayed tumor development in vivo and decreased the rate of cell growth in vitro. These results suggest that Egr-1 plays an oncogenic role in HER2/neu-driven mammary tumorigenesis.     

Combination of Quercetin and 2-Methoxyestradiol Enhances Inhibition of Human Prostate Cancer LNCaP and PC-3 Cells Xenograft Tumor Growth

Quercetin and 2-Methoxyestradiol (2-ME) are promising anti-cancer substances. Our previous in vitro study showed that quercetin synergized with 2-Methoxyestradiol exhibiting increased antiproliferative and proapoptotic activity in both androgen-dependent LNCaP and androgen-independent PC-3 human prostate cancer cell lines. In the present study, we determined whether their combination could inhibit LNCaP and PC-3 xenograft tumor growth in vivo and explored the underlying mechanism. Human prostate cancer LNCaP and PC-3 cells were inoculated subcutaneously in male BALB/c nude mice. When xenograft tumors reached about 100 mm³, mice were randomly allocated to vehicle control, quercetin or 2-Methoxyestradiol singly treated and combination treatment groups. After therapeutic intervention for 4 weeks, combination treatment of quercetin and 2-ME i) significantly inhibited prostate cancer xenograft tumor growth by 46.8% for LNCaP and 51.3% for PC-3 as compared to vehicle control group, more effective than quercetin (28.4% for LNCaP, 24.8% for PC3) or 2-ME (32.1% for LNCaP, 28.9% for PC3) alone; ii) was well tolerated by BALB/c mice and no obvious toxic reactions were observed; iii) led to higher Bax/Bcl-2 ratio, cleaved caspase-3 protein expression and apoptosis rate; and iv) resulted in lower phosphorylated AKT (pAKT) protein level, vascular endothelial growth factor protein and mRNA expression, microvascular density and proliferation rate than single drug treatment. These effects were more remarkable compared to vehicle group. Therefore, combination of quercetin and 2-ME can serve as a novel clinical treatment regimen owning the potential of enhancing antitumor effect on prostate cancer in vivo and lessening the dose and side effects of either quercetin or 2-ME alone. These in vivo results will lay a further solid basis for subsequent researches on this novel therapeutic regimen in human prostate cancer.     

TUSC1, a Putative Tumor Suppressor Gene,Reduces Tumor Cell Growth In Vitro and Tumor Growth In Vivo

We previously reported the identification of TUSC1 (Tumor Suppressor Candidate 1), as a novel intronless gene isolated from a region of homozygous deletion at D9S126 on chromosome 9p in human lung cancer. In this study, we examine the differential expression of TUSC1 in human lung cancer cell lines by western blot and in a primary human lung cancer tissue microarray by immunohistochemical analysis. We also tested the functional activities and mechanisms of TUSC1 as a tumor suppressor gene through growth suppression in vitro and in vivo. The results showed no expression of TUSC1 in TUSC1 homozygously deleted cells and diminished expression in some tumor cell lines without TUSC1 deletion. Interestingly, the results from a primary human lung cancer tissue microarray suggested that higher expression of TUSC1 was correlated with increased survival times for lung cancer patients. Our data demonstrated that growth curves of tumor cell lines transfected with TUSC1 grew slower in vitro than those transfected with the empty vector. More importantly, xenograph tumors in nude mice grew significantly slower in vivo in cells stably transfected with TUSC1 than those transfected with empty vector. In addition, results from confocal microscopy and immunohistochemical analyses show distribution of TUSC1 in the cytoplasm and nucleus in tumor cell lines and in normal and tumor cells in the lung cancer tissue microarray. Taken together, our results support TUSC1 has tumor suppressor activity as a candidate tumor suppressor gene located on chromosome 9p.     

Prospectively Isolated Cancer-Associated CD10+ Fibroblasts Have Stronger Interactions with CD133+ Colon Cancer Cells than with CD133? Cancer Cells

Although CD133 has been reported to be a promising colon cancer stem cell marker, the biological functions of CD133⁺ colon cancer cells remain controversial. In the present study, we investigated the biological differences between CD133⁺ and CD133⁻ colon cancer cells, with a particular focus on their interactions with cancer-associated fibroblasts, especially CD10⁺ fibroblasts. We used 19 primary colon cancer tissues, 30 primary cultures of fibroblasts derived from colon cancer tissues and 6 colon cancer cell lines. We isolated CD133⁺ and CD133⁻ subpopulations from the colon cancer tissues and cultured cells. In vitro analyses revealed that the two populations showed similar biological behaviors in their proliferation and chemosensitivity. In vivo analyses revealed that CD133⁺ cells showed significantly greater tumor growth than CD133⁻ cells (P = 0.007). Moreover, in cocultures with primary fibroblasts derived from colon cancer tissues, CD133⁺ cells exhibited significantly more invasive behaviors than CD133⁻ cells (P<0.001), especially in cocultures with CD10⁺ fibroblasts (P<0.0001). Further in vivo analyses revealed that CD10⁺ fibroblasts enhanced the tumor growth of CD133⁺ cells significantly more than CD10⁻ fibroblasts (P<0.05). These data demonstrate that the in vitro invasive properties and in vivo tumor growth of CD133⁺ colon cancer cells are enhanced in the presence of specific cancer-associated fibroblasts, CD10⁺ fibroblasts, suggesting that the interactions between these specific cell populations have important roles in cancer progression. Therefore, these specific interactions may be promising targets for new colon cancer therapies.     

Cytotoxic capacity of a novel glycosylated antitumor ether lipid in chemotherapy-resistant high grade serous ovarian cancer in vitro and in vivo

Chemotherapy resistant high grade serous ovarian cancer remains a clinically intractable disease with a high rate of mortality. We tested a novel glycosylated antitumor ether lipid called l-Rham to assess the in vitro and in vivo efficacy on high grade serous ovarian cancer cell lines and patient samples. l-Rham effectively kills high grade serous ovarian cancer cells grown as 2D or 3D cultures in a dose and time dependent manner. l-Rham efficacy was tested in vivo in a chicken allantoic membrane/COV362 xenograft model, where l-Rham activity was as effective as paclitaxel in reducing tumor weight and metastasis. The efficacy of l-Rham to reduce OVCAR3 tumor xenografts in NRG mice was assessed in low and high tumor burden models. l-Rham effectively reduced tumor formation in the low tumor burden group, and blocked ascites formation in low and high tumor burden animals. l-Rham demonstrates efficacy against OVCAR3 tumor and ascites formation in vivo in NRG mice, laying the foundation for further development of this drug class for the treatment of high grade serous ovarian cancer patients.