ASPP and iASPP: Implication in cancer development and progression.

The well-known guardian of genome, p53 plays critical roles in the induction of apoptosis typically upon DNA damage whereas mutant p53 containing cells are unable to undergo apoptosis which leads to aggressive tumor growth and drug resistance. Moreover, another molecule regulating wild-ype p53 function is ASPP (apoptosis stimulating proteins of p53) family. ASPP family consists of ASPP1 and ASPP2, and functions as tumor suppressors whereas the inhibitor of ASPP (iASPP) functions as oncogene. By binding to apoptosis regulating proteins such as p53, p63, p73, Bcl-2, NF-κB p65, etc., ASPP1 and ASPP2 promote apoptosis while overexpression of iASPP inhibits apoptotic cell death typically after DNA damage. In cancer cells, the aberrant expressions of ASPP1, ASPP2 and iASPP have been observed, especially, the high expression of iASPP in cancers is associated with worse disease status, therapy resistance and poor survival of patients with cancers. The molecular interactions between the members of ASPP family and their binding proteins in apoptotic pathway together with other regulators such as miR-124, NF-κB regulated Twist, snail, etc. form a complex signal transduction network to control apoptosis and tumor growth. Therefore, targeting ASPP family could regulate the aberrant communications in the signal transduction network to induce apoptosis and drug sensitivity. Several peptides, miRNAs and natural agents have been used to target ASPP family and show encouraging results in the induction of apoptosis of cancer cells; however, more in vivo animal studies and clinical trials are needed to confirm the true value of targeting ASPP family in the treatment of cancers.

MicroRNAs in personalized cancer therapy.

MicroRNAs (miRNAs) are small endogenous noncoding single-stranded RNAs. They critically regulate the post-transcriptional activity of several key physiological and pathological cell processes including cancer. Through their transcriptional regulatory functions, miRNAs control tumor proliferation, invasion and metastasis. The expression of miRNAs is altered in malignancies. It could be either upregulated or downregulated depending upon the role of a particular miRNA in the pathogenetic development of the tumor. The upregulated miRNAs exert an 'oncogenic' effect leading to tumor proliferation and metastasis. The downregulated miRNAs have 'tumor suppressor' effects. Recent studies have demonstrated that miRNAs have a role in the early diagnosis, prognosis and treatment outcome assessment of cancers. Every tumor has specific miRNA alterations, i.e. some are overexpressed and others are downregulated. These altered miRNAs can be used as a tumor-specific 'signature' for potential clinical use in improving the accuracy of diagnosis, determining prognosis and as therapeutic targets for therapy. Specific miRNAs can be targeted using oligonucleotide sequences corresponding to the altered miRNAs. These are referred to as 'antagomirs'. Depending upon the miRNA alterations in the tumor of an individual patient, one could design targeted therapies for personalized medicine in patients. Hence, miRNAs have an immense role in personalized cancer therapy.

MicroRNA profiling of diagnostic needle aspirates from patients with pancreatic cancer.

BACKGROUND: A major challenge to the development of biomarkers for pancreatic cancer (PC) is the small amount of tissue obtained at the time of diagnosis. Single-gene analyses may not reliably predict biology of PC because of its complex molecular makeup. MicroRNA (miRNA) profiling may provide a more informative molecular interrogation of tumours. The primary objective of this study was to determine the feasibility of performing miRNA arrays and quantitative real-time PCR (qRT-PCR) from archival formalin-fixed paraffin-embedded (FFPE) cell blocks obtained from fine-needle aspirates (FNAs) that is the commonest diagnostic procedure for suspected PC. METHODS: MicroRNA expression profiling was performed on FFPE from FNA of suspicious pancreatic masses. Subjects included those who had a pathological diagnosis of pancreatic adenocarcinoma and others with a non-malignant pancreatic histology. Exiqon assay was used to quantify miRNA levels and qRT-PCR was used to validate abnormal expression of selected miRNAs. RESULTS: A total of 29 and 15 subjects had pancreatic adenocarcinoma and no evidence of cancer, respectively. The RNA yields per patient varied from 25 to 100 ng. Profiling demonstrated deregulation of over 228 miRNAs in pancreatic adenocarcinoma of which the top 7 were further validated by qRT-PCR. The expression of let-7c, let-7f, and miR-200c were significantly reduced in most patients whereas the expression of miR-486-5p and miR-451 were significantly elevated in all pancreas cancer patients. MicroRNAs let-7d and miR-423-5p was either downregulated or upregulated with a significant inter-individual variation in their expression. CONCLUSION: This study demonstrated the feasibility of using archival FFPE cell blocks from FNAs to establish RNA-based molecular signatures unique to pancreatic adenocarcinoma with potential applications in clinical trials for risk stratification, patient selection, and target validation.

Pancreatic cancer stem cells and EMT in drug resistance and metastasis.

Pancreatic cancer (PC) is an aggressive malignancy with one of the worst outcomes among all cancers. It is the fourth leading cause of cancer death in the United States with a very low five-year survival rate. The high mortality of PC could, in part, be due to their drug resistance characteristics and high propensity for metastasis. Recently, cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT)-type cells, which shares molecular characteristics with CSCs, have been believed to play critical roles in drug resistance and cancer metastasis as demonstrated in several human malignancies including PC. Thus, the discovery of molecular knowledge of drug resistance and metastasis in relation to CSCs and EMT in PC is becoming an important area of research, and such knowledge is likely to be helpful in the discovery of newer drugs as well as designing novel therapeutic strategies for the treatment of PC with better outcome. In this brief review, we will summarize the current knowledge regarding the CSCs and EMT in the context of drug resistance and metastasis in PC, the molecular events occurring in CSCs and EMT, and the design of novel therapeutic strategies targeting CSCs and EMT-type cells to increase drug sensitivity and suppression of metastasis toward better treatment outcome of patients diagnosed with PC.

Antiintegrin alpha v beta 3 blocks human breast cancer growth and angiogenesis in human skin.

Angiogenesis plays a fundamental role in human breast tumor progression. In fact, recent findings indicate that vascular density is a prognostic indicator of breast cancer disease status. Evidence is presented that the integrin alpha v beta 3 is not only a marker of human breast tumor-associated blood vessels, but that it plays a significant role in human angiogenesis and breast tumor growth. To assess the role of alpha v beta 3-dependent angiogenesis in the progression of human breast cancer, we examined a SCID mouse/human chimeric model with transplanted full thickness human skin containing alpha v beta 3-negative human breast tumor cells. This tumor induced a human angiogenic response as measured by vascular cell immunoreactivity with monoclonal antibodies LM609 and P2B1 directed to human alpha v beta 3 and CD31, respectively. Intravenous administration of LM609 either prevented tumor growth or markedly reduced tumor cell proliferation within the microenvironment of the human skin. These LM609-treated tumors not only contained significantly fewer human blood vessels but also appeared considerably less invasive than tumors in control animals. These findings demonstrate that alpha v beta 3 antagonists may provide an effective antiangiogenic approach for the treatment of human breast cancer.

Reduction of QM protein expression correlates with tumor grade in prostatic adenocarcinoma.

The QM protein is a transcription cofactor inhibiting the activity of AP-1 transcription factors and is also a ribosomal protein participating in protein synthesis. While protein synthesis is known to be increased in many cancers, inhibition of AP-1 activity presumably suppresses development and growth of sex-hormone-regulated tumor cells. The present study is the first report on immunohistochemical data of QM in human prostatic tissues. Paraffin sections of human prostate cancer samples were immunohistochemically stained for QM. The staining scores were analyzed with the clinicopathologic data of the patients. QM protein expression was found in all normal prostate glands adjacent to prostate cancer and in various intraepithelial neoplasia (PIN). In prostate cancer, the staining intensity and stained areas were decreased, compared to the normal glands and PIN lesions; in high-grade tumors only some patches of tumor cells showed positivity. Intense (3+) staining was mostly observed in the Gleason grade three areas (48%) compared to grade 4 and 5 areas (22%), although both low and high-grade tumors showed similar percentages of weakly stained areas. Moreover, staining in prostatic adenocarcinoma was often topographically patchy and varied from negative or weak (1+) to intense (3+). There was an inverse correlation from normal to low-grade tumors and then to high-grade tumors. However, in high-grade tumors, the positive areas were mostly confined to peripheral aspects of tumors and were particularly strong in foci of perineural invasion. This preliminary study suggests that decreased QM expression may be associated with early development of prostate cancer, but later a high level of QM may facilitate progression of the tumors to a more aggressive phenotype.

p53 status and prognosis of locally advanced prostatic adenocarcinoma: a study based on RTOG 8610.

BACKGROUND: The p53 tumor suppressor gene (also known as TP53) is one of the most frequently mutated genes in human cancer. Several studies have shown that p53 mutations are infrequent in prostate cancer and are associated with advanced disease. PURPOSE: We assessed the prognostic value of identifying abnormal p53 protein expression in the tumors of patients with locally advanced prostate cancer who were treated with either external-beam radiation therapy alone or total androgen blockade before and during the radiation therapy. METHODS: The study population consisted of a subset of patients entered in Radiation Therapy Oncology Group protocol 8610 ("a phase III trial of Zoladex and flutamide used as cytoreductive agents in locally advanced carcinoma of the prostate treated with definitive radiotherapy"). Immunohistochemical detection of abnormal p53 protein in pretreatment specimens (i.e., needle biopsies or transurethral resections) was achieved by use of the monoclonal anti-p53 antibody DO7; specimens in which 20% or more of the tumor cell nuclei showed positive immunoreactivity were considered to have abnormal p53 protein expression. Associations between p53 protein expression status and the time to local progression, the incidence of distant metastases, progression-free survival, and overall survival were evaluated in univariate (logrank test) and multivariate (Cox proportional hazards model) analyses. Reported P values are two-sided. RESULTS: One hundred twenty-nine (27%) of the 471 patients entered in the trial had sufficient tumor material for analysis. Abnormal p53 protein expression was detected in the tumors of 23 (18%) of these 129 patients. Statistically significant associations were found between the presence of abnormal p53 protein expression and increased incidence of distant metastases (P = .04), decreased progression-free survival (P = .03), and decreased overall survival (P = .02); no association was found between abnormal p53 protein expression and the time to local progression (P = .58). These results were independent of the Gleason score and clinical stage. A significant treatment interaction was detected with respect to the development of distant metastases: Among patients receiving both radiation therapy and hormone therapy, those with tumors exhibiting abnormal p53 protein expression experienced a reduced time to the development of distant metastases (P = .001); for patients treated with radiation therapy alone, the time to distant metastases was unrelated to p53 protein expression status (P = .91). CONCLUSIONS: Determination of p53 protein expression status yield significant, independent prognostic information concerning the development of distant metastases, progression-free survival, and overall survival for patients with locally advanced prostate cancer who are treated primarily with radiation therapy. IMPLICATIONS: The interaction of radiation therapy plus hormone therapy and abnormal p53 protein expression may provide a clinical link to experimental evidence that radiation therapy and/or hormone therapy act, at least in part, by the induction of apoptosis (a cell death program) and suggests that this mechanism may be blocked in patients whose tumors have p53 mutations.

Growth of human nondiploid primary prostate tumor epithelial cells in vitro.

Research into molecular and cellular defects underlying prostate cancer would be advanced by in vitro models of prostate tumor cells representing patient tumors. We have propagated, in serum-free medium, epithelial cell cultures derived from nondiploid prostate tumors and normal human prostate. The serial passage tumor cells exhibited nondiploid karyotype and transformed phenotypes of focus formation and anchorage-independent growth. In contrast, the normal prostate cells showed diploid karyotype and lacked transformed phenotypes. Both the tumor and normal cells were positive for prostate-specific antigen and cytokeratins 18 and 19 and negative for keratin 15. These results demonstrate that the nondiploid prostate tumors and normal prostate epithelial cell cultures retained their respective in vivo properties and should allow studies to elucidate molecular alterations involved in human prostate cancer.

bcl-2 suppresses expression of p21WAF1/CIP1 in breast epithelial cells.

The tumor suppressor gene p53 regulates G1 checkpoint prior to the initiation of DNA synthesis, which can either induce G1 arrest or signal apoptosis. The involvement of p53 in apoptosis may also be related to its ability to down-regulate transcription of the bcl-2 gene. The bcl-2 gene product prevents most types of apoptotic cell death, suggesting that bcl-2 interferes with an essential signaling molecule involved in the apoptotic cell death pathway. Although the bcl-2 protein is shown to be overexpressed in many types of human tumor including breast cancer, its biochemical or pathological consequences are poorly understood. To determine the effects of bcl-2 overexpression on apoptosis and transformation of breast epithelial cells and to investigate whether bcl-2 interferes with the p53 pathway, we introduced the bcl-2 expression vector into MCF10A cells, which were derived from diploid human breast epithelial cells containing the wild-type p53 gene. Overexpression of bcl-2 prevented free radical-induced apoptosis and induced a partially transformed phenotype in MCF10A cells. Although overexpression of bcl-2 did not affect the expression of the p53 gene, p53-dependent gene transcription such as p21WAF1/CIP1 was suppressed. These results suggest that bcl-2 may inhibit p53 functional activity and is involved in the regulation of an early commitment step either to proliferate or suicide.

Induction of apoptosis in breast cancer cells by TPA.

Bcl-2, Bax and p53 gene products have been linked to programmed cell death pathways. p21WAF1 has been shown to mediate p53-induced cell cycle arrest and to inhibit cyclin-dependent kinase activity. We have analysed the expression of these genes and apoptosis induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in several human breast cancer cell line. We found up-regulation of p21WAF1 and Bax expressions, however, the expressions of p53 and Bcl-2 genes remained unchanged in TPA-treated cells. Furthermore, DNA ladder formation and PARP cleavage were observed after treatment for 24 h, indicating apoptotic cell death. Flow cytometry with 7-amino actinomycin D staining showed that the number of apoptotic cells increased with longer treatment of TPA. From these results, we conclude that TPA is not only a tumor promoter, but also induces apoptosis in breast cancer cells. TPA-induced apoptosis appears to be mediated through a p53-independent pathway, and the up-regulation of p21WAF1 and Bax may be the molecular mechanisms by which TPA induces apoptosis.

Induction of apoptosis in breast cancer cells MDA-MB-231 by genistein.

Breast cancer is the most common cancer among American women, whereas Asian women, who consume a traditional diet high in soy products, have a relatively low incidence. Genistein is a prominent isoflavonoid in soy products and has been proposed as the agent responsible for lowering the rate of breast cancer in Asian women. We investigated the effects of genistein on cell growth and apoptosis-related gene expression in breast cancer cells MDA-MB-231. We found up-regulation of Bax and p21WAF1 expressions and down-regulation of Bcl-2 and p53 expression in genistein-treated cells. Furthermore, DNA ladder formation, CPP32 activation, and PARP cleavage were observed after treatment with genistein, indicating apoptotic cell deaths. Flow cytometry with 7-amino actinomycin D staining showed that the number of apoptotic cells increased with longer treatment of genistein. From these results, we conclude that genistein inhibits the growth of MDA-MB-231 breast cancer cells, regulates the expression of apoptosis-related genes, and induces apoptosis through a p53-independent pathway. The up-regulation of Bax and p21WAF1 may be the molecular mechanisms by which genistein induces apoptosis, however, further definitive studies are needed. These results suggest that genistein may be a potentially effective chemopreventive or therapeutic agent against breast cancer.

Indole-3-carbinol (I3C) induced cell growth inhibition, G1 cell cycle arrest and apoptosis in prostate cancer cells.

Prostate cancer is one of the most common cancers in men and it is the second leading cause of cancer related death in men in the United States. Recent dietary and epidemiological studies have suggested the benefit of dietary intake of fruits and vegetables in lowering the incidence of prostate cancer. A diet rich in fruits and vegetables provides phytochemicals, particularly indole-3-carbinol (I3C), which may be responsible for the prevention of many types of cancer, including hormone-related cancers such as prostate. Studies to elucidate the role and the molecular mechanism(s) of action of I3C in prostate cancer, however, have not been conducted. In the current study, we investigated whether I3C had any effect against prostate cancer cells and, if so, attempts were made to identify the potential molecular mechanism(s) by which I3C elicits its biological effects on prostate cancer cells. Here we report for the first time that I3C inhibits the growth of PC-3 prostate cancer cells. Induction of G1 cell cycle arrest was also observed in PC-3 cells treated with I3C, which may be due to the observed effects of I3C in the up-regulation of p21(WAF1) and p27(Kip1) CDK inhibitors, followed by their association with cyclin D1 and E and down-regulation of CDK6 protein kinase levels and activity. The induction of p21(WAF1) appears to be transcriptionally upregulated and independent of the p53 responsive element. In addition, I3C inhibited the hyperpohosphorylation of the Retinoblastoma (Rb) protein in PC-3 cells. Induction of apoptosis was also observed in this cell line when treated with I3C, as measured by DNA laddering and poly (ADP-ribose) polymersae (PARP) cleavage. We also found an up-regulation of Bax, and down-regulation of Bcl-2 in I3C-treated cells. These effects may also be mediated by the down-regulation of NF-kappaB observed in I3C treated PC-3 cells. From these results, we conclude that I3C inhibits the growth of PC-3 prostate cancer cells by inducing G1 cell cycle arrest leading to apoptosis, and regulates the expression of apoptosis-related genes. These findings suggest that I3C may be an effective chemopreventive or therapeutic agent against prostate cancer.

Translocation of Bax to mitochondria induces apoptotic cell death in indole-3-carbinol (I3C) treated breast cancer cells.

Epidemiological studies have suggested that the consumption of fruits and vegetables that provide several classes of compounds, including Indole-3-carbinol (I3C), may have chemopreventive activity against breast cancer. Several in vitro and in vivo animal studies also provide convincing evidence for the anti-tumor activity of I3C, however, the molecular mechanism(s) by which I3C exerts its biological effects on breast cancer cells has not been fully elucidated. In this study, we investigated the effects of I3C in Her-2/neu over-expressing MDA-MB-435 breast cancer cells and compared these results with parental cells transfected with control vector. We focused our investigation in elucidating the molecular mechanism(s) by which I3C induces apoptosis in breast cancer cells. Our data show that I3C inhibits breast cancer cell growth in a dose dependent manner in Her-2/neu over-expressing and in normal Her-2/neu expressing cells. Induction of apoptosis was also observed in these cell lines when treated with I3C, as measured by poly (ADPribose) polymerase (PARP) and caspase-3 activation. In addition, we found that I3C up-regulates Bax, down-regulates Bcl-2 and, thereby, increased the ratio of Bax to Bcl-2 favoring apoptosis. These results suggest that the alteration in the expression of these genes may play an important role in mediating the biological effects of I3C. Moreover, we also show the cellular localization of Bax by confocal microscopy, which showed diffuse distribution of Bax throughout the cytoplasmic compartment in breast cancer cells in control culture. However, in I3C treated cells, Bax showed a punctate pattern of distribution that was localized in the mitochondria. From these results, we conclude that the over-expression and translocation of Bax to mitochondria causes mitochondrial depolarization and activation of caspases, which may be one of the mechanism(s) by which I3C induces apoptotic processes in I3C treated breast cancer cells. Overall, our present data provide a novel molecular mechanism(s) by which I3C elicits its biological effects on both Her-2/neu over-expressing and with normal Her-2/neu expressing breast cancer cells, suggesting that I3C could be an effective agent in inducing apoptosis in breast cancer cells.

Ciprofloxacin mediated cell growth inhibition, S/G2-M cell cycle arrest, and apoptosis in a human transitional cell carcinoma of the bladder cell line.

The second most prevalent urological malignancy in middle aged and elderly men is bladder cancer, with 90% of the cases being transitional cell carcinomas. The success of current systemic and intravesical therapeutic agents, such as cisplatin, thiotepa, Adriamycin, mitomycin C, and bacillus Calmette-Guerin, is limited with recurrence rates reduced to 17-44%. In addition, most of these agents require instrumentation of the urinary tract and are delivered at a significant cost and potential morbidity to the patient. Fluroquinolone antibiotics such as ciprofloxacin, which can be administered p.o., may have a profound effect in bladder cancer management. This is primarily based on limited in vitro studies on tumor cells derived from transitional cell carcinoma of the bladder that revealed a dose- and time-dependent inhibition of cell growth by ciprofloxacin at concentrations that are easily attainable in the urine of patients. However, the mechanism(s) by which ciprofloxacin elicits its biological effects on bladder cancer cells is not well documented. Our experimental data confirm previous studies showing the in vitro cell growth inhibition of the transitional cell carcinoma of the bladder cell line HTB9 and further showed the induction of cell cycle arrest at the S/G2-M checkpoints. In addition, we found down-regulation of cyclin B, cyclin E, and dephosphorylation of cdk2 in ciprofloxacin-treated bladder tumor cells. There was also an up-regulation of Bax, which altered the Bax:Bcl-2 ratio, which may be responsible for mitochondrial depolarization reported to be involved prior to the induction of apoptosis. The cyclin-dependent kinase inhibitor p21WAF1 level was found to be decreased within 12 h of ciprofloxacin treatment and disappeared completely when HTB9 cells were treated with 200 microg/ml ciprofloxacin for 24 h. The down-regulation of p21WAF1 closely correlated with poly(ADP-ribose) polymerase cleavage and CPP32 activation. Recent studies revealed that p21WAF1 protects cells from apoptosis by arresting them in G1 and further binds to pro-caspase-3, preventing its activation and thus, inhibiting the apoptotic cascade. Hence, the down-regulation of p21WAF1, together with the alterations in Bax and cdk2 as observed in our studies, may define a novel mechanism by which ciprofloxacin inhibits tumor cell growth and induces apoptotic cell death. The results of our current studies provide strong experimental evidence for the use of ciprofloxacin as a potential preventive and/or therapeutic agent for the management of transitional cell carcinoma of the bladder.

Genistein elicits pleiotropic molecular effects on head and neck cancer cells.

Genistein (4,5,7-trihydroxyisoflavone) has been reported to induce cell cycle arrest and apoptosis in different cancer cell lines in vitro and to show antitumor activity against a variety of tumors in animal models. We have previously reported (S. A. Alhasan et al., Nutr. Cancer, 34:12-19, 1999; S. A. Alhasan et al., Int. J. Oncol., 16: 333-338, 2000) that genistein induces cell cycle arrest and apoptosis by up-regulating p21(WAF1) and Bax, and down-regulating cyclin B1 and Bcl-2 in a head and neck cancer cell line. However, the precise molecular mechanism(s) by which genistein elicits its effects on head and neck cancer cells still remains to be elucidated. In the present study, we report that genistein induces several specific molecular changes in head and neck cancer cells, such as down-regulation of c-erbB-2 expression, down-regulation of MMP-2 and MMP-9 secretion, inhibition of tumor cell invasion and down-regulation of nuclear factor-kappaB DNA binding activity. In addition, genistein inhibited the levels of phosphorylated Akt and the expression of 14-3-3 protein. Moreover, genistein induces telomere shortening in treated cells without affecting telomerase activity in vitro. We also observed that genistein inhibits the translocation of telomerase catalytic subunit [human telomerase reverse transcriptase (hTERT)] to the nucleus, which may result in telomere shortening, although the activity of telomerase is unaffected, along with the inhibition of metaphase spread of chromosomes. From these results, together with our previously published reports, (S. A. Alhasan et al., Nutr. Cancer, 34: 12-19, 1999; S. A. Alhasan et al., Int. J. Oncol., 16: 333-338, 2000) we conclude that genistein elicits pleiotropic molecular changes that resulting in the inhibition of cell growth and the induction of apoptotic cell death of head and neck cancer cells, which suggests that genistein may be useful as a chemotherapeutic and/or chemopreventive agent for head and neck cancer.

Differential sensitivity of normal and malignant breast epithelial cells to genistein is partly mediated by p21(WAF1).

Genistein, a soy metabolite, is a potential chemopreventive agent against various types of cancer. There are several studies documenting molecular alterations leading to cell cycle arrest and induction of apoptosis in a variety of cancer cells; however, no studies, to date, have shown the effect of genistein in isogenic normal and malignant breast epithelial cells. In this study, we investigated whether genistein shows any differential sensitivity to normal (MCF10A and MCF12A) and malignant (MCF10CA1a and MDA-MB-231) breast epithelial cells. We found that genistein causes a greater degree of G(2)-M arrest and induces apoptosis in malignant cell lines compared with normal breast epithelial cells. After genistein treatment, flow cytometric analysis revealed a hyperdiploid population in malignant cells that was not observed in normal cells. Cell cycle regulator p21(WAF1), which is known to be up-regulated by genistein treatment, was greatly induced at RNA and protein levels in normal cells, whereas its level was only slightly induced in malignant MDA-MB-231 cells and not detectable in malignant MCF10CA1a cells. Therefore, we investigated the causal role of p21(WAF1) in the differential sensitivity of genistein among these cell lines. We examined the effects of genistein on p21(WAF1) -/- and p21(WAF1) +/+ HCT116 cells, which were used as controls prior to studies on breast cancer cells. We found that there was a greater degree of cell cycle arrest and apoptosis in p21(WAF1) -/- cells compared with p21(WAF1) +/+ HCT116 cells after genistein treatment. Flow cytometric analysis after genistein treatment showed a significant number of p21(WAF1) -/- cells in the hyperdiploid population, which are probably programmed to die through apoptotic processes. To further confirm the causal role of p21(WAF1) in genistein-mediated cell cycle arrest and apoptosis, we down-regulated p21(WAF1) by antisense p21(WAF1) cDNA transfection experiments. We found that both normal and malignant p21(WAF1) antisense (AS)-expressing clones became more sensitive to G(2)-M arrest after genistein treatment. Flow cytometric analysis showed an increase in the hyperdiploid population in the AS clones. Further evaluation showed an increase in apoptosis in malignant AS clones but not in normal breast epithelial AS clones. These results suggest that p21(WAF1) may play an important role in determining the sensitivity of normal and malignant breast epithelial cells to genistein.

Induction of apoptosis and inhibition of c-erbB-2 in breast cancer cells by flavopiridol.

Flavopiridol is a flavone that inhibits several cyclin-dependent kinases and exhibits potent growth-inhibitory activity against a number of human tumor cell lines, both in vitro and when grown as xenografts in mice. It is presently being investigated as a novel antineoplastic agent in the primary screen conducted by the Developmental Therapeutics Program, National Cancer Institute. Because breast cancer is the most common cancer and second leading cause of cancer-related deaths in women in the United States, we investigated whether flavopiridol could be an effective agent against a series of isogenic breast- cancer cell lines having different levels of erbB-2 expression and differential invasion and metastatic characteristics. Flavopiridol was found to inhibit the growth of MDA-MB-435 (parental) and 435.eB (stable transfectants) cells that were established by transfecting c-erbB-2 cDNA into MDA-MB-435. Induction of apoptosis was also observed in these cell lines when treated with flavopiridol, as measured by DNA laddering, PARP, and CPP32 cleavages. We also found modest up-regulation of Bax and down-regulation of Bcl-2, but there was a significant down-regulation of c-erbB-2 in flavopiridol-treated cells. Gelatin zymography showed that flavopiridol inhibits the secretion of matrix metalloproteinase (MMP; MMPs 2 and 9) in the breast cancer cells and that the inhibition of c-erbB-2 and MMPs may be responsible for the inhibition of cell invasion observed in flavopiridol-treated cells. Collectively, these molecular effects of flavopiridol, however, were found to be independent of c-erbB-2 overexpression, suggesting that flavopiridol may be effective in all breast cancer. From these results, we conclude that flavopiridol inhibits the growth of MDA-MB-435 breast cancer cells, induces apoptosis, regulates the expression of genes, and inhibits invasion and, thus, may inhibit metastasis of breast cancer cells. These findings suggest that flavopiridol may be an effective chemotherapeutic or preventive agent against breast cancer.

Genistein potentiates the radiation effect on prostate carcinoma cells.

We have shown previously that genistein, the major isoflavone in soybean, inhibited the growth of human prostate cancer cells in vitro by affecting the cell cycle and inducing apoptosis. To augment the effect of radiation for prostate carcinoma, we have now tested the combination of genistein with photon and neutron radiation on prostate carcinoma cells in vitro. The effects of photon or neutron radiation alone or genistein alone or both combined were evaluated on DNA synthesis, cell growth, and cell ability to form colonies. We found that neutrons were more effective than photons for the killing of prostate carcinoma cells in vitro, resulting in a relative biological effectiveness of 2.6 when compared with photons. Genistein at 15 microM caused a significant inhibition in DNA synthesis, cell growth, and colony formation in the range of 40-60% and potentiated the effect of low doses of 200-300 cGy photon or 100-150 cGy neutron radiation. The effect of the combined treatment was more pronounced than with genistein or radiation alone. Our data indicate that genistein combined with radiation inhibits DNA synthesis, resulting in inhibition of cell division and growth. Genistein can augment the effect of neutrons at doses approximately 2-fold lower than photon doses required to observe the same efficacy. These studies suggest a potential of combining genistein with radiation for the treatment of localized prostate carcinoma.

An orthotopic model of human pancreatic cancer in severe combined immunodeficient mice: potential application for preclinical studies.

Pancreatic adenocarcinoma is one of the most incurable and least understood of all human cancers. It is the fourth leading cause of cancer-related mortality in males (after lung, prostate, and colon) and in females (after lung, breast, and colon) in the United States with <2-3% of patients surviving >5 years. In an attempt to search for more effective therapies for this disease, we report here, for the first time, an effective treatment, the combination of gemcitabine and auristatin-phenethylamine (PE), against an orthotopic implantation of a human pancreatic adenocarcinoma cell line (HPAC) in severe combined immunodeficient (SCID) mice. Tumor implantation was performed by injecting 100 microl of the HPAC cell suspension (1 x 10(6) cells) directly into the pancreas of 5-week-old SCID mice. After implantation, tumor formation was checked twice a week. All palpable tumors were detected within 21 days (100% take rate), and tumors were confirmed histologically to be pancreatic adenocarcinoma. For the subsequent efficacy trial, tumor-bearing SCID mice were randomized into four groups with five mice in each group. One served as a control, the second received gemcitabine alone (2.5 mg/kg/injection i.p.), the third received auristatin-PE alone (2.0 mg/kg/injection i.v.), and the fourth group received the combination of gemcitabine (i.p.) and auristatin-PE (1.5 mg/kg/injection i.v.). All animals were euthanized 7 days after the completion of their treatments, and the pancreases were resected. Histological examination revealed the tumors to be adenocarcinoma. The tumors were composed of diffuse sheets of cells interrupted by glandular spaces containing secretory material. Cytologically, the tumor cells were large, pleomorphic, and hyperchromatic. Many cells contained intracellular lumina containing mucin. Immunohistochemical studies showed strong p21WAF1 (p21) expression but no immunoreactivity with p53 and Her-2/neu antibodies. The mean pancreatic weight in the gemcitabine/auristatin-PE combination group was significantly (P = 0.014) lower (0.84 +/- 0.639 g) when compared with those of the control (2.91 +/- 1.19 g) and gemcitabine alone (1.84 +/- 0.796 g; P = 0.064) groups. In addition, the mean weight in the combination group approached statistical significance when compared with the auristatin-PE group alone (1.16 +/- 0.635 g; P = 0.028). We conclude that the combination of gemcitabine and auristatin-PE is an effective treatment against HPAC tumors in this xenograft model and more effective than treatment with either gemcitabine or auristatin-PE alone and could be considered for future animal studies with pancreas cancer and/or for human clinical trials.

Sex-independent synthesis of alpha 2u-globulin and its messenger ribonucleic acid in the rat preputial gland: biochemical and immunocytochemical analyses.

alpha 2u-Globulin is the principal urinary protein of the mature male rat. The major urinary source of this protein is the liver where it is synthesized and secreted by hepatocytes under hormonal regulation. High levels of alpha 2u-globulin and its messenger RNA (mRNA) are also present in the preputial gland of both male and female rats, and neither castration nor ovariectomy significantly alters the preputial concentration of this protein and its mRNA. Per unit mass of RNA and protein, the preputial gland as compared to liver contains about 3-fold higher level of alpha 2u-globulin mRNA and about 300-fold higher level of alpha 2u-globulin. Despite a 3-fold (300%) difference in the content of alpha 2u-globulin mRNA, nuclear run-off experiments show only a 30% higher rate of alpha 2u-globulin gene transcription in the preputial gland than in the liver. Immunocytochemical analyses reveal that the liver possesses two alpha 2u-globulin cell populations, one showing higher immunoreactivity than the other. In contrast, the preputial gland contains only one type of alpha 2u-globulin containing acinar cells, and a large amount of alpha 2u-globulin accumulates in the ductal lumen. From these results we conclude that the 300% higher level of alpha 2u-globulin mRNA in the preputial gland is not due to a corresponding difference in the rate of transcription of alpha 2u-globulin gene. Such a difference may represent tissue-specific regulation at a posttranscriptional level of mRNA metabolism. Furthermore, the huge difference in the alpha 2u-globulin content of the preputial gland and the liver is primarily due to the cellular and ductal accumulation of this protein in the preputial gland vs. its rapid secretion by the liver.