Cation-dependent mannose-6-phosphate receptor expression and distribution are influenced by estradiol in MCF-7 breast cancer cells.

Cancer cells secrete procathepsin D, and its secretion is enhanced by estradiol. Although alterations in the pro-enzyme intracellular transport have been reported, the mechanism by which it is secreted remains poorly understood. In this work, we have studied the influence of estradiol on the expression and distribution of the cation-dependent mannose-6-phosphate receptor (CD-MPR), which would be a key molecule to ensure the proper localization of the enzyme to lysosomes in breast cancer cells. Immunoblotting studies demonstrated that the expression of CD-MPR is higher in MCF-7 cells, as compared to other breast cancer and non-tumorigenic cells. This expression correlated with high levels of cathepsin D (CatD) in these cells. By immunofluorescence, this receptor mostly co-localized with a Golgi marker in all cell types, exhibiting an additional peripheral labelling in MCF-7 cells. In addition, CD-MPR showed great differences regarding to cation-independent mannose-6-phosphate receptor. On the other hand, the treatment with estradiol induced an increase in CD-MPR and CatD expression and a re-distribution of both proteins towards the cell periphery. These effects were blocked by the anti-estrogen tamoxifen. Moreover, a re-distribution of CD-MPR to plasma membrane-enriched fractions, analyzed by gradient centrifugation, was observed after estradiol treatment. We conclude that, in hormone-responsive breast cancer cells, CD-MPR and CatD are distributed together, and that their expression and distribution are influenced by estradiol. These findings strongly support the involvement of the CD-MPR in the pro-enzyme transport in MCF-7 cells, suggesting the participation of this receptor in the procathepsin D secretion previously reported in breast cancer cells.

Methylation profile of triple-negative breast carcinomas.

Breast cancer is a group of clinically, histopathologically and molecularly heterogeneous diseases, with different outcomes and responses to treatment. Triple-negative (TN) breast cancers are defined as tumors that lack the expression of estrogen receptor, progesterone receptor and epidermal growth factor receptor 2. This subgroup accounts for 15% of all types of breast cancer and its prevalence is higher among young African, African-American and Latino women. The hypermethylation of CpG islands (CpGI) is a common epigenetic alteration for suppressing gene expression in breast cancer and has been shown to be a key factor in breast carcinogenesis. In this study we analyzed the hypermethylation of 110 CpGI within 69 cancer-related genes in TN tumors. For the methylation analysis, we used the methyl-specific multiplex-ligation probe amplification assay. We found that the number of methylated CpGI is similar between TN and non-TN tumors, but the methylated genes between the groups are different. The methylation profile of TN tumors is defined by the methylation of five genes (that is, CDKN2B, CD44, MGMT, RB and p73) plus the non-methylation of 11 genes (that is, GSTP1, PMS2, MSH2, MLH1, MSH3, MSH6, DLC1, CACNA1A, CACNA1G, TWIST1 and ID4). We conclude that TN tumors have a specific methylation profile. Our findings give new information for better understanding tumor etiology and encourage future studies on potential drug targets for triple-negative breast tumors, which now lack a specific treatment.

Predictive value of apoptosis, proliferation, HER-2, and topoisomerase IIalpha for anthracycline chemotherapy in locally advanced breast cancer.

PURPOSE: Laboratory evidence indicates that tumor growth depends on the balance between cell proliferation and cell death, and many anticancer agents may exert their therapeutic effect by decreasing proliferation and increasing apoptosis. Additionally, clinical observations indicate that overexpression of HER-2 or topoisomerase IIalpha (topo IIalpha) may be predictors of better response to anthracyclines in breast cancer. The objective of this study was to determine if proliferation (Ki-67), apoptosis (TUNEL), and expression of HER-2 and topo IIalpha are affected by anthracycline treatment, and if these molecular markers predict anthracycline responsiveness. EXPERIMENTAL DESIGN: Thirty-three women with primary breast tumors > or =3 cm received either doxorubicin (75 mg/m(2)) or epirubicin (120 mg/m(2)) for 4 cycles before surgery. Clinical response was evaluated after 4 cycles of treatment. Changes in molecular markers were assessed from core needle taken before treatment (D0), at 24-48 h (Dl) and on day 7 (D7) while on treatment, and from the surgical specimen excised on day 84 (D84) after the fourth cycle of chemotherapy. RESULTS: The overall response rate was 51% (17 of 33 patients), with a 12% complete clinical response rate (4 of 33 patients). There were trends for tumors with higher apoptosis and topo IIalpha at baseline (D0) to be more responsive to anthracyclines, p = 0.1 and p = 0.08, respectively. Median apoptosis increased from D0 to Dl (p = 0.06) while median Ki-67 decreased (p = 0.07). Overall, expression of HER-2 remained stable throughout the chemotherapy administration. By Day 84, topo IIalpha had significantly decreased from baseline in responders, while it increased in non-responders, p = 0.03. CONCLUSIONS: In human primary breast cancer, anthracycline treatment causes an early increase in apoptosis and a decrease in proliferation. In this pilot study, higher apoptosis and topo IIalphaa levels in primary tumors were associated with greater responsiveness to anthracyclines, and topo IIalpha levels declined in responsive tumors.

A silver staining method for single-cell gel assay.

The single-cell gel assay (comet assay) is a very useful microelectrophoretic technique for evaluation of DNA damage and repair in individual cells. Usually, the comets are visualized and evaluated with fluorescent DNA stains. This staining requires specific equipment (e.g., a high-quality fluorescence microscope), the slides must be analyzed immediately, and they cannot be stored for long periods of time. Here we describe, using human lymphocytes, some modifications of the silver staining for comets that significantly increase the sensitivity/reproducibility of the assay. This silver staining was compared with fluorescence staining and commercial silver stains. (J Histochem Cytochem 49:1183-1186, 2001)

Marcadores moleculares en pacientes con cáncer de cuello uterino tratadas con quimioterapia neoadyuvante y radioterapia / Molecular markers in patients with cervical cancer treated with induction chemotherapy and radiation therapy

Objetivos: En este estudio prospectivo de determinaron las modificaciones en la expresión y el valor predictivo de p53, p21 wafi/sdII/cipi, PCNA, hMLH1, hMSH2, Bcl'2 y TUNEL en pacientes con cáncer de cervix localmente avanzado tratadas con quimioterapia de inducción y radioterapia. Pacientes y métodos: Se obtuvieron muestras de 24 pacientes (IB'bulky/IIIB, 95 por ciento carcinomas escamosos) antes de la quimioterapia y a los 30 días del tratamiento. Trece pacientes recibieron un esquema de drogas basado en cisplatino y como la respuesta a esta terapia no fue buena, a las otras 11 pacientes se les administró vinorelbine e ifosfamida. Luego de la quimioterapia todas las pacientes recibieron radioterapia. La expresión de los marcadores moleculares en las biopsias pre- y post quimioterapia se estudió por inmunohistoquímica y la apoptosis fue evaluada por la técnica del TUNEL mejorada recientemente. Para comparar los cambios en la expresión de los marcadores moleculares y para correlacionarlos con la evaluación clínica (media de seguimiento: 31 meses para las pacientes que recibieron cisplatino y 19 para las que recibieron vinorelbine e ifosfamida) se realizaron análisis estadísticos. Resultados y conclusiones: La quimioterapia de inducción no aumentó la sobrevida de las pacientes, el 50 por ciento tuvo enfermedad progresiva (EP) o falleció (F). La expresión de p21waf1/sdII/cip1, hMLF1, hMSH2, y Bcl-2 no mostró cambios significativos después de la quimioterapia y no correlacionó con la evaluación clínica. La expresión de p53 no se modificó luego de la quimioterapia, las pacientes con tumores p53 positivos mostraron una tendencia a tener una sobrevida menor. Las pacientes con EP o que fallecieron mostraron niveles altos de PCNA, a diferencia de aquellas que estuvieron libres de enfermedad (LE) o con enfermedad estable (EE) (50 por ciento versus 17 por ciento, respectivamente, p<0.004). La sobrevida de las pacientes con bajos índices de TUNEL (igual o menor al valor medio entre las biopsias pre y post-quimioterapia de 1.5) fue significativamente más corta que las pacientes que presentaron índices de TUNEL altos (p<0.009). Nuestros resultados muestran que la quimioterapia de inducción (los dos tratamientos aplicados en este estudio) no mejoró la sobrevida de pacientes con cáncer de cervix...

Marcadores moleculares en pacientes con cáncer de cuello uterino tratadas con quimioterapia neoadyuvante y radioterapia / Molecular markers in patients with cervical cancer treated with induction chemotherapy and radiation therapy

Objetivos: En este estudio prospectivo de determinaron las modificaciones en la expresión y el valor predictivo de p53, p21 wafi/sdII/cipi, PCNA, hMLH1, hMSH2, Bcl2 y TUNEL en pacientes con cáncer de cervix localmente avanzado tratadas con quimioterapia de inducción y radioterapia. Pacientes y métodos: Se obtuvieron muestras de 24 pacientes (IBbulky/IIIB, 95 por ciento carcinomas escamosos) antes de la quimioterapia y a los 30 días del tratamiento. Trece pacientes recibieron un esquema de drogas basado en cisplatino y como la respuesta a esta terapia no fue buena, a las otras 11 pacientes se les administró vinorelbine e ifosfamida. Luego de la quimioterapia todas las pacientes recibieron radioterapia. La expresión de los marcadores moleculares en las biopsias pre- y post quimioterapia se estudió por inmunohistoquímica y la apoptosis fue evaluada por la técnica del TUNEL mejorada recientemente. Para comparar los cambios en la expresión de los marcadores moleculares y para correlacionarlos con la evaluación clínica (media de seguimiento: 31 meses para las pacientes que recibieron cisplatino y 19 para las que recibieron vinorelbine e ifosfamida) se realizaron análisis estadísticos. Resultados y conclusiones: La quimioterapia de inducción no aumentó la sobrevida de las pacientes, el 50 por ciento tuvo enfermedad progresiva (EP) o falleció (F). La expresión de p21waf1/sdII/cip1, hMLF1, hMSH2, y Bcl-2 no mostró cambios significativos después de la quimioterapia y no correlacionó con la evaluación clínica. La expresión de p53 no se modificó luego de la quimioterapia, las pacientes con tumores p53 positivos mostraron una tendencia a tener una sobrevida menor. Las pacientes con EP o que fallecieron mostraron niveles altos de PCNA, a diferencia de aquellas que estuvieron libres de enfermedad (LE) o con enfermedad estable (EE) (50 por ciento versus 17 por ciento, respectivamente, p<0.004). La sobrevida de las pacientes con bajos índices de TUNEL (igual o menor al valor medio entre las biopsias pre y post-quimioterapia de 1.5) fue significativamente más corta que las pacientes que presentaron índices de TUNEL altos (p<0.009). Nuestros resultados muestran que la quimioterapia de inducción (los dos tratamientos aplicados en este estudio) no mejoró la sobrevida de pacientes con cáncer de cervix... (AU)

Differential anthracycline sensitivity in two related human colon carcinoma cell lines expressing similar levels of P-glycoprotein.

Chemosensitivity of the human colon carcinoma HCT-15 cell line to 4'-epidoxorubicin proved to be 100-fold higher than that of its variant HCT-15 EDR. Confocal scanning microscopy showed significant less drug accumulation in HCT-15 EDR. A 2-fold increase in hsp27 expression was found in HCT-15 EDR, with no alteration in hsp70. The expression of the drug exporter Pgp was similar in both cell lines, despite the lower drug accumulation shown by HCT-15 EDR in respect to HCT-15. Other molecules implicated in the acquisition of enhanced chemoresistance or a more active Pgp variant present in HCT-15 EDR, could explain the phenomenon.

c-erbB-2 (HER-2/neu) protein and drug resistance in breast cancer patients treated with induction chemotherapy.

Expression of c-erbB-2 protein has been associated with poor prognosis and poor response to chemotherapy in breast cancer patients. In the present prospective study, we have analyzed whether c-erbB-2, p53 and P170 proteins may be determinants of tumor resistance in locally advanced breast cancer patients treated with induction chemotherapy. Biopsies (n = 60) were examined by immuno-histochemistry; in 62% of cases core or incisional biopsies were taken before drug administration, allowing comparison in paired biopsies of the cytological and molecular changes induced by treatment Sixty percent of the patients received relatively high doses of FAC or FEC (5-fluorouracil, doxorubicin or epirubicin and cyclophosphamide), and 40% received relatively high doses of doxorubicin or epirubicin alone. No significant changes were observed in the molecular markers studied following chemotherapy; in the few biopsies where changes appeared, the changes did not exhibit any significant or similar trend. For 30 of the patients who received FAC/FEC treatment, follow-up reached a median of 34 months. In these cases, neither the clinical (reduction in tumor size) nor the histological (evaluated after neoadjuvant chemotherapy) responses showed statistically significant differences between the patients who developed distant metastases and the disease-free patients. c-erbB-2 was over-expressed in 50% of patients who developed distant metastases vs. 7% of the disease-free patients. Disease free survival (DFS) curves between c-erbB-2-positive and c-erbB-2-negative patients were statistically significant. No correlation between p53 or P170 expression with DFS was found. Our results suggest that c-erbB-2 protein expression is associated with development of distant metastases in breast cancer patients treated with relatively high doses of anthracyclines in induction chemotherapy.

Heat shock protein expression and drug resistance in breast cancer patients treated with induction chemotherapy.

Heat shock proteins (Hsps) are induced in vitro by several cytotoxic drugs; in human breast cancer cells these proteins appear to be involved in anti-cancer drug resistance. The present report was designed to analyze whether chemotherapy affects in vivo the expression of Hsp27, Hsp70, Hsc70 and Hsp90 in breast cancer patients treated with induction chemotherapy and whether these proteins may be determinants of tumor resistance to drug administration. We have analyzed 35 biopsies from breast cancer patients treated with induction chemotherapy. Expression of the Hsps in the tumors was compared with (i) histological and clinical responses to chemotherapy, (ii) tumor cell proliferation measured by proliferating cell nuclear antigen (PCNA) immunostaining and nucleolar organizer regions (AgNORs) staining and (iii) the expression of estrogen and progesterone receptors. We also compared disease-free survival (DFS) and overall survival (OS) with the expression of the Hsps studied. After chemotherapy, nuclear Hsp27 and Hsp70 expression was increased and Hsp70 and Hsc70 cytoplasmic expression was decreased. A high nuclear proportion of Hsp70 in tumor cells (>10%) correlated significantly with drug resistance. We also observed that patients whose tumors expressed nuclear or a high cytoplasmic proportion (>66%) of Hsp27 had shorter DFS. The combination of Hsp27 and Hsp70 levels showed a strong correlation with DFS. Neither the cellular proliferation nor the levels of steroid receptors showed any significant difference before or after drug administration or during follow-up of patients. Our results suggest that Hsp27 and Hsp70 are involved in drug resistance in breast cancer patients treated with combination chemotherapies.

Heat shock proteins and cell proliferation in human breast cancer biopsy samples.

Human breast cancers may overexpress certain heat shock protein (hsp) family members, proteins which are involved with cell proliferation and differentiation as well as with disease prognosis and drug resistance. Here, we have studied the relationship between the expression of two hsps (hsp27 and hsp70) and the proliferative activity of tumor cells in 40 biopsies from breast cancer patients. Twenty of these tumors were selected for a detailed colocalization study. Immunocytochemistry was done using specific antibodies against hsp27 and hsp70. Cell proliferation was studied analyzing the expression of proliferating cell nuclear antigen (PCNA) (late G1, S, and G2 phases of the cell cycle) and the number of silver-staining nucleolar organizer regions (AgNORs) (G1 phase). The colocalization study revealed a statistically significant inverse correlation between hsp27 expression and cell proliferation in 16/19 (84%) of the cases evaluated by PCNA immunostaining, and in 11/16 (69%) of the cases evaluated by AgNORs. In contrast, a statistically significant positive correlation between hsp70 expression and elevated cell proliferation was seen in almost 85% of the cases evaluated by PCNA staining, and in almost 50% of the cases evaluated by AgNORs. Moreover, in 22% (9/40) of the breast cancer samples examined, hsp70 was clearly associated with the mitotic spindle. A Western blot analysis revealed that hsp70 was coprecipitated with taxol-polymerized tubulin. The association of hsp70 with the mitotic spindle was not clearly noted in lung carcinoma samples (N = 20) or in normal cells displaying elevated mitotic activity. These studies thus demonstrate that in a significant percentage of clinical breast cancers hsp27 overexpression is inversely correlated with cell proliferation, while hsp70 is clearly associated with the mitotic spindle and cell proliferation. These results add evidence to the concept that in human breast cancers hsp27 may be involved in cell growth arrest and increased differentiation while, in contrast, hsp70 may be involved in cell proliferation; further studies will be necessary to elucidate these possible cause-and-effect relationships.

Estrogen receptors, progesterone receptors, and cell proliferation in human breast cancer.

The breast is a target organ for estrogens and progesterone. These hormones control several functions of the normal and abnormal mammary epithelium including cell proliferation. Most of the actions of estrogens and progesterone are mediated via specific steroid receptors, and one would expect that proliferating cells should contain estrogen receptors (ER) and/or progesterone receptors (PR). However, the correlation between receptor expression and cell proliferation is still controversial. In the present study we have examined 29 human breast cancer samples; in 17 of them we evaluated the simultaneous ER and PR localization with that of proliferating cell nuclear antigen (PCNA) and silver-stained nucleolar organizer regions (AgNORs) in a cell-by-cell study. We found that in almost 50% of the tumor biopsies examined, the cells expressing ER were significantly associated with elevated cell proliferation. In another group (38%) there were not significant differences between ER expression and cell proliferation. In only one of the samples (6%) the cells expressing ER showed lower cell proliferation. The study also revealed that in 44% of the tumors the PR expressing cells were associated with elevated cell proliferation. In a second group the PR expression was not significantly associated with cell proliferation (33% of the cases). Finally, in 22% of the samples the cells carrying PR showed lower cell proliferation. We also detected lower ER immunoreactivity in 30% of the breast cancer biopsies with one of the monoclonal antibodies against ER (antibody 1D5 directed against the A/B domain). This group of tumors was PR-negative (or very weakly positive) and had high proliferation. The presence of tumors with 'abnormal' ER proteins and displaying ER/PR significantly associated with elevated cell proliferation could have implications in human breast cancer treatment.

Effects of short-term tamoxifen administration in patients with invasive cervical carcinoma.

Cervical cancer is not considered a hormone-responsive tumor in spite of the presence of estrogen receptors (ER) and progesterone receptors (PgR) in some of them. Endocrine treatments have not achieved clinical responses, however, tamoxifen has been reported to induce PgR and to inhibit cell growth of many cervical carcinoma cell lines. In this study we investigated whether tamoxifen administration affects the histopathological characteristics of cervical cancer and the expression of ER, PgR, HER-2/neu and p53 protein. Nineteen patients with invasive cervical cancer free of previous treatments were studied. The triphenylethylene antiestrogen tamoxifen was given orally during 10 days (20 or 40 mg/day). Pre- and post-tamoxifen biopsies were evaluated using slides stained with hematoxylin and eosin and immunostained (ER, PgR, HER-2/neu, p53, PCNA, keratin, heat shock protein 27,000 daltons). Estrogen receptors were present in 37% and PgR in 16% of the biopsies from untreated patients. Only one case that was PgR-negative before tamoxifen administration showed weak PgR-positivity following antiestrogen administration. No obvious changes were observed in ER, HER-2/neu and p53 proteins. A statistically significant decrease in the number of mitotic figures was obtained in 16% (3/19) of the post-tamoxifen biopsies and two of them showed higher differentiation. The results showed that tamoxifen did not induce changes in estrogen-regulated proteins in cervical cancer. However, the data showed that certain cervical carcinomas had changes in their proliferation and differentiation levels following tamoxifen administration. These findings suggest that tamoxifen may affect some cervical cancer tissues by a hormone-independent mechanism(s).