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Agents that induce apoptosis in breast cancer cells have great potential to facilitate chemotherapeutic intervention and improve patient outcomes. In this study, the effects of injecting purified human chorionic gonadotropin (hCG) directly into human breast cancer xenografts grown in nude mice were examined. It was shown that intratumoral injection of purified hCG increased the apoptotic index in breast cancer xenografts. These results were supported by the findings that exposure of breast cancer cells to purified hCG decreased cell viability in five different breast cancer cell lines. In some of these cell lines, the effects of hCG in cell viability appear to correlate with activation/expression of the hCG/luteinizing hormone receptor. Preoperative apoptotic induction by factors such as purified hCG may improve local control or work synergistically with neoadjuvant chemotherapy to improve complete pathologic response of locally advanced breast cancer.

Resveratrol is a naturally occurring phytoalexin with antioxidant and antiinflammatory properties. Recent studies suggest that resveratrol possesses anticancer effects, although its mechanism of action is not well understood. We now show that resveratrol inhibits Src tyrosine kinase activity and thereby blocks constitutive signal transducer and activator of transcription 3 (Stat3) protein activation in malignant cells. Analyses of resveratrol-treated malignant cells harboring constitutively-active Stat3 reveal irreversible cell cycle arrest of v-Src-transformed mouse fibroblasts (NIH3T3/v-Src), human breast (MDA-MB-231), pancreatic (Panc-1), and prostate carcinoma (DU145) cell lines at the G0-G1 phase or at the S phase of human breast cancer (MDA-MB-468) and pancreatic cancer (Colo-357) cells, and loss of viability due to apoptosis. By contrast, cells treated with resveratrol, but lacking aberrant Stat3 activity, show reversible growth arrest and minimal loss of viability. Moreover, in malignant cells harboring constitutively-active Stat3, including human prostate cancer DU145 cells and v-Src-transformed mouse fibroblasts (NIH3T3/v-Src), resveratrol treatment represses Stat3-regulated cyclin D1 as well as Bcl-xL and Mcl-1 genes, suggesting that the antitumor cell activity of resveratrol is in part due to the blockade of Stat3-mediated dysregulation of growth and survival pathways. Our study is among the first to identify Src-Stat3 signaling as a target of resveratrol, further defining the mechanism of antitumor cell activity of resveratrol and raising its potential application in tumors with an activated Stat3 profile.

Colon cancer overexpresses insulin-like growth factor 1 (IGF1) and insulin-like growth factor 1 receptor (IGF1-R), as compared with normal or adenomatous mucosa, and it has been postulated that colorectal cancer cells depend on the IGF1/IGF1-R pathway for their growth and progression. In this study, using the human colon cancer cell line HCT116, we find that established HCT116/IGF1-R transfectants exhibit a more aggressive transformed phenotype than the parental cell line, as demonstrated by their higher proliferation rate in response to IGF1, higher degree of anchorage-independent growth, resistance to serum deprivation-induced apoptosis, and higher migratory capability in a monolayer "wounding assay." When injected into nude mice, HCT116/IGF1-R transfectants were highly invasive and produced distant metastases, whereas the parental cell did not. Moreover, the overexpression of IGF1-R in these cells was associated with IGF1-R-induced activation of Akt and up-regulation of the antiapoptotic protein Bcl-x(L). We also show that Akt pathway mediates IGF1-R-induced Bcl-x(L) expression at transcriptional level. Our data demonstrate, for the first time, that IGF1-R/Akt/Bcl-x(L) pathway may contribute to a more aggressive malignant phenotype, in a subset of colorectal cancers.

Colorectal carcinomas have been found to express increased levels of IGF1 and IGF1-R, as compared to normal or adenomatous colonic mucosa, and it has been postulated that a subset of colorectal cancers are under the autocrine regulation of the IGF1/IGF1-R system. In this study, we selected human colorectal carcinoma cell lines with high (SW620, HT29, L4A) and low (CaCo2, and HCT 116) expression of IGF1-R by flow cytometry. Compared to the IGF1-R(-) cells, the IGF1-R(+) cells revealed a more aggressive phenotype as demonstrated by a higher proliferation rate (approximately 2-fold increase) in response to IGF1, higher degree of transformation (approximately 5-to-15-fold increase in colony formation in soft agar), increased resistance to serum deprivation-induced apoptosis [1-7 apoptotic cells/5 microscopic fields, as compared to 37 to 101 apoptotic cells/5 microscopic fields of the IGF1-R(-) cells], and higher migratory capability measured by a wounding assay [IGF1-R(+) cells migrated a distance of up to 15 millimeters from the cut edge of the monolayer, while the IGF1-R(-) cells were able to migrate only 2-3 millimeters away from the same reference point]. While the cell lines overexpressing the IGF1-R had higher levels of Src activation, the use of a Src inhibitor reduced the IGF1-R protein expression, slowed down the proliferation of IGF1-R(+) cells, and reduced their colony formation in soft agar. Based on the above observations, we conclude that an overexpressed and activated IGF1-R may increase the degree of transformation and motility of colon cancer cells by activating c-Src.

Signal transducers and activators of transcription (STATs) are transcription factors that mediate normal biologic responses to cytokines and growth factors. However, abnormal activation of certain STAT family members, including Stat3, is increasingly associated with oncogenesis. In fibroblasts expressing the Src oncoprotein, activation of Stat3 induces specific gene expression and is required for cell transformation. Although the Src tyrosine kinase induces constitutive Stat3 phosphorylation on tyrosine, activation of Stat3-mediated gene regulation requires both tyrosine and serine phosphorylation of Stat3. We investigated the signaling pathways underlying the constitutive Stat3 activation in Src oncogenesis. Expression of Ras or Rac1 dominant negative protein blocks Stat3-mediated gene regulation induced by Src in a manner consistent with dependence on p38 and c-Jun N-terminal kinase (JNK). Both of these serine/threonine kinases and Stat3 serine phosphorylation are constitutively induced in Src-transformed fibroblasts. Furthermore, inhibition of p38 and JNK activities suppresses constitutive Stat3 serine phosphorylation and Stat3-mediated gene regulation. In vitro kinase assays with purified full-length Stat3 as the substrate show that both JNK and p38 can phosphorylate Stat3 on serine. Moreover, inhibition of p38 activity and thus of Stat3 serine phosphorylation results in suppression of transformation by v-Src but not v-Ras, consistent with a requirement for Stat3 serine phosphorylation in Src transformation. Our results demonstrate that Ras- and Rac1-mediated p38 and JNK signals are required for Stat3 transcriptional activity induced by the Src oncoprotein. These findings delineate a network of tyrosine and serine/threonine kinase signaling pathways that converge on Stat3 in the context of oncogenesis.

The antioxidant N-acetyl-L-cysteine (NAC) has been increasingly used as an experimental tool to assess involvement of reactive oxygen species in cell signaling and is being evaluated as a preventive and therapeutic agent for cancer and pulmonary diseases related to inflammation and oxidative stress. However, a detailed characterization of the effect of NAC on cell cycle progression has not been reported. In the present study, modulation of cell cycle progression by NAC was analyzed in mouse fibroblast NIH3T3 cells grown in 10% fetal bovine serum. Complete inhibition of NIH3T3 cell proliferation was obtained with 20 mM NAC. Inhibition of cell proliferation by NAC (at or below 20 mM) was not due to cell death, and the antiproliferative effect of NAC was reversible. Flow cytometric analysis of cell cycle phase distribution indicated that NAC blocked the cell cycle in the G1 phase. Consistent with this observation, NAC inhibited DNA synthesis. After releasing the G1-block by NAC, S phase re-entry occurred between 8 and 12 h, suggesting that NAC blocked the cell cycle in early to mid-G1 phase. NAC prevented activation of mitogen-activated protein (MAP) kinases p42MAPK and p44MAPK and inhibited expression of cyclin D1, but had no effect on the levels of proliferating cell nuclear antigen. Incubation of cells with PD98059, a specific inhibitor of MAP kinase kinase 1, partially arrested the cell cycle in the G1 phase. These results indicate that the antiproliferative effect of NAC is linked in part to inhibition of the MAP kinase pathway.

Nitrogen dioxide (NO2) is a well-known environmental air toxin, produced from a variety of sources, including cigarette smoke. Because of the growing knowledge of the harmful effects of passive smoking on children, we decided to study the effect of NO2 exposure on the release of surfactant from isolated neonatal type II pulmonary epithelial cells. After isolation from 1 to 4 day old rabbits, type II epithelial cells were allowed to adhere for 18 hours, washed, media changed, and were exposed to either 5% CO2 in room air or NO2, 5 ppm, for 2 hours (all results mean +/- sd; comparisons, paired t-test). There was no difference in cell number or viability prior to exposure. Cells exposed to NO2 had an increase in LDH release [LDH activity in media/(LDH in media+cells) x 100], air 12.6 +/- 2.2%, NO2 21.7 +/- 3.7%, (p < 0.05). NO2-exposed cells also had an increase in total phospholipid (microgram/cell culture dish) in media compared to air exposed, air 170.13 +/- 7.54, NO2 195.15 +/- 11.2, (p < 0.05). 3H-choline incorporation as a precursor to disaturated phosphatidylcholine (DSPC) was also conducted during exposure to either air or NO2. Incorporation of 3H-choline into surfactant lipid was increased in media from cells after NO2 exposure compared to air, 58.23 +/- 15.16 air, 76.81 +/- 19.86 NO2 (cpm/microgram protein; p < 0.05). These results show that 2 hours of 5 ppm NO2 exposure is associated with an increase in release of surfactant from neonatal type II cells in culture.