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Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. Five-fluorouracil (5FU) remains the single most effective treatment for advanced disease, despite a response rate of only 20%. Herein, we show that the antioxidants pyrrolidinedithiocarbamate and vitamin E induce apoptosis in CRC cells. This effect is mediated by induction of p21WAF1/CIP1, a powerful inhibitor of the cell cycle, through a mechanism involving C/EBPbeta (a member of the CCAAT/enhancer binding protein family of transcription factors), independent of p53. Antioxidants significantly enhance CRC tumor growth inhibition by cytotoxic chemotherapy in vitro (5FU and doxorubicin) and in vivo (5FU). Thus, chemotherapeutic agents administered in the presence of antioxidants may provide a novel therapy for colorectal cancer.
The Fas/Fas ligand (FasL) pathway is widely involved in apoptotic cell death in lymphoid and nonlymphoid cells. It has recently been postulated that many chemotherapeutic agents also induce cell death by activating the Fas/FasL pathway. In the present study we compared apoptotic pathways induced by anti-Fas or chemotherapeutic agents in the Jurkat human T-cell leukemia line. Immunoblotting showed that treatment of wild-type Jurkat cells with anti-Fas or the topoisomerase II-directed agent etoposide resulted in proteolytic cleavage of precursors for the cysteine-dependent aspartate-directed proteases caspase-3 and caspase-7 and degradation of the caspase substrates poly(ADP-ribose) polymerase (PARP) and lamin B1. Likewise, affinity labeling with N-(N(alpha)-benzyloxycarbonylglutamyl-N(epsilon)-biotinyllysyl+ ++)aspartic acid [(2,6-dimethyl-benzoyl)oxy]methyl ketone [Z-EK(bio)D-amok] labeled the same five active caspase species after each treatment, suggesting that the same downstream apoptotic pathways have been activated by anti-Fas and etoposide. Treatment with ZB4, an antibody that inhibits Fas-mediated cell death, failed to block etoposide-induced apoptosis, raising the possibility that etoposide does not initiate apoptosis through Fas/FasL interactions. To further explore the relationship between Fas- and chemotherapy-induced apoptosis, Fas-resistant Jurkat cells were treated with various chemotherapeutic agents. Multiple independently derived Fas-resistant Jurkat lines underwent apoptosis that was indistinguishable from that of the Fas-sensitive parental cells after treatment with etoposide, doxorubicin, topotecan, cisplatin, methotrexate, staurosporine, or gamma-irradiation. These results indicate that antineoplastic treatments induce apoptosis through a Fas-independent pathway even though Fas- and chemotherapy-induced pathways converge on common downstream apoptotic effector molecules.
It is proposed that loss of a growth-inhibitory response to transforming growth factor beta (TGFbeta) contributes to breast cancer progression. Because cellular TGFbeta responsiveness often correlates with TGFbeta type II receptor (TGFbeta-IIR) expression, we have examined the cellular distribution of TGFbeta-IIRs in tumor and nontumor mammary epithelial cells. By immunoblot analysis, TGFbeta-IIR was detected both in membrane and cytosolic fractions of MDA-231 tumor cells as well as in normal human breast epithelial cells. The cytosolic protein appeared to be more abundant and was detected as a clear perinuclear staining by immunocytochemistry. The glycosylation patterns of the cytosolic and membrane form were different, indicating distinct receptor pools. The cytosolic TGFbeta-IIR did not bind 125I-labeled TGFbeta1 but had a detectable in vitro and in vivo kinase activity. MCF-7 breast cancer cells express the TGFbeta-IIR mRNA but show undetectable cell surface TGFbeta-IIR protein by affinity cross-linking. However, low levels of TGFbeta-IIR were observed in MCF-7 cytosol. Sequencing of the coding region of TGFbeta-IIR from MCF-7 cells indicated a point mutation (A439V) in a nonconserved region of the kinase domain. When MCF-7 cells were treated with sublethal doses of Adriamycin that induce cell differentiation, the membrane localization of TGFbeta-IIR and TGFbeta response were restored. Our results indicate the presence of a prominent, kinase-active TGFbeta-IIR in the cytosol of several mammary cell lines. This cytosolic pool of receptors is the only detectable one in MCF-7 cells. Loss of wild-type membrane receptors due to defects in trafficking presents a potential new mechanism for escape from negative growth control.
Mutations in the retinoblastoma (pRb) tumor suppressor pathway including its cyclin-cdk regulatory kinases, or cdk inhibitors, are a hallmark of most cancers and allow unrestrained E2F-1 transcription factor activity, which leads to unregulated G1-to-S-phase cell cycle progression. Moderate levels of E2F-1 overexpression are tolerated in interleukin 3 (IL-3)-dependent 32D.3 myeloid progenitor cells, yet this induces apoptosis when these cells are deprived of IL-3. However, when E2F activity is augmented by coexpression of its heterodimeric partner, DP-1, the effects of survival factors are abrogated. To determine whether enforced E2F-1 expression selectively sensitizes cells to cytotoxic agents, we examined the effects of chemotherapeutic agents and radiation used in cancer therapy. E2F-1 overexpression in the myeloid cells preferentially sensitized cells to apoptosis when they were treated with the topoisomerase II inhibitor etoposide. Although E2F-1 alone induces moderate levels of p53 and treatment with drugs markedly increased p53, the deleterious effects of etoposide in E2F-1-overexpressing cells were independent of p53 accumulation. Coexpression of Bcl-2 and E2F-1 in 32D.3 cells protected them from etoposide-mediated apoptosis. However, Bcl-2 also prevented apoptosis of these cells upon exposure to 5-fluorouracil and doxorubicin, which were also cytotoxic for control cells. Pretreating E2F-1-expressing cells with ICRF-193, a second topoisomerase II inhibitor that does not damage DNA, protected the cells from etoposide-induced apoptosis. However, ICRF-193 cooperated with DNA-damaging agents to induce apoptosis. Therefore, topoisomerase II inhibition and DNA damage can cooperate to selectively induce p53-independent apoptosis in cells that have unregulated E2F-1 activity resulting from mutations in the pRb pathway.
PURPOSE - The curative resection rate in patients with potentially resectable carcinoma of the esophagus is approximately 55% and their median survival time is 11 months. Preoperative chemotherapy with high doses of chemotherapeutic agents was used to evaluate clinical and pathologic responses, curative resection rate, toxicity, and survival. Colony-stimulating factor (CSF) was added to reduce the severity of myelosuppression.
PATIENTS AND METHODS - Twenty-six consecutive assessable patients with potentially resectable adenocarcinoma of the esophagus or gastroesophageal junction were treated with two preoperative courses of intensive chemotherapy (etoposide, doxorubicin, and cisplatin [EAP]) with granulocyte-macrophage CSF (GM-CSF). Additional three conventional-dose postoperative chemotherapy courses without GM-CSF were given to patients who responded to preoperative chemotherapy.
RESULTS - A median of three courses (range, one to six), were administered. Of 27 patients, 26 were assessable for response to preoperative EAP; 13 (50%) achieved a major response. Among 23 patients who underwent surgery, 15 (65%) had a curative resection (58% of 26 assessable patients); none of the patients had a pathologic complete response, but two patients had only microscopic carcinoma in the resected specimen. Six patients had carcinoma present at the resection margins and received postoperative radiotherapy. Two patients were found to have liver metastases at exploration. At a median follow-up of 22 months, the median survival of 26 patients was 12.5 months (range, 2 to 32 +). Fourteen patients died of their carcinoma; two patients died of treatment-related causes; one died of an unrelated CNS arterial malformation; and the causes of death in two patients remain unknown. Seven patients are alive with no evidence of relapse. Major toxicities of this regimen included severe myelosuppression, nausea and vomiting, infections, and severe constitutional symptoms related to GM-CSF. However, subcutaneous injection of GM-CSF was well tolerated.
CONCLUSION - High-dose EAP is active against locoregional adenocarcinoma of the esophagus and gastroesophageal junction but can be associated with significant toxicity. Although this strategy remains attractive and needs to be developed further, less toxic and more effective regimens need to be identified.
BACKGROUND - The authors retrospectively analyzed 87 patients with malignant thymoma treated at M.D. Anderson Cancer Center between 1951 and 1990. The analysis examined the clinical stages, histologic types, and treatment modalities and attempted to determine if chemotherapy had an impact on survival.
METHODS - The patients were divided into three groups by their year of treatment and treatment modality. Patients treated between 1951 and 1975 were in Group I; patients treated between 1976 and 1980 were in Group II; and patients treated between 1981 and 1990 were in Group III. Most of the patients (18 [72%] in Group I; 16 [62%] in Group II; and 18 [50%] in Group III) had surgical resection alone or with radiotherapy. Patients with advanced-stage disease in Group I received single-agent chemotherapy, whereas patients with advanced-stage disease in Group II received a different, combination chemotherapy regimen, and those in Group III were treated primarily with cisplatin- and doxorubicin-based combination chemotherapy, e.g., the cyclophosphamide doxorubicin, and cisplatin with or without prednisone. The 17 patients treated with cisplatin with or without prednisone were separately evaluated for survival according to their response.
RESULTS - Twenty-eight patients (5 [20%] in Group I; 6 [23%] in Group II; and 17 [47%] in Group III) received chemotherapy alone or after surgery or radiotherapy. The cisplatin with or without prednisone regimen was used in 17 Group III patients for initial treatment or for relapse. The overall response rate among the patients receiving the cisplatin with or without prednisone regimen was 64%; 6 (35%) had a complete response, and 5 (29%) had a partial response. Thirty-one (36%) of the 87 total patients had 45 recurrent tumors; the lung (29%), pleura (22%), and mediastinum (18%) were the most common sites of recurrence, whereas bone was the most common distant metastatic site. The 5-year survival rate was 70% in patients with Stage I disease, 71% in patients with Stage II or III disease, and 46% in patients with Stage IV disease. The 10-year survival rate was 70% in patients with Stage I disease, 47% in patients with Stage II or III disease, and 21% in patients with Stage IV disease. Statistical analysis indicated a significant difference among the survival rates of patients with noninvasive Stage I, invasive Stage II plus III (P = 0.033), and Stage II plus III and IV tumors (P = 0.056), but not between patients with Stage II or III tumors. Patients with a major response to the cisplatin with or without prednisone regimen had a significant survival improvement compared to those with no response (P = 0.002, log-rank test).
CONCLUSIONS - Because thymoma is a chemosensitive tumor and frequently recurs in patients with Stage II or greater disease, chemotherapy carries a potential survival benefit and should be incorporated into the multimodality approach to prolong disease-free survival.
The treatment of advanced metastatic prostate cancer by hormone manipulation or orchiectomy is frequently followed by the appearance of hormone-insensitive and highly chemoresistant tumor cells. In this study we have investigated the contribution of the P-glycoprotein-mediated drug efflux (multidrug-resistance; MDR) to the cellular resistance of prostate carcinoma-derived cell lines to diverse cytotoxic drugs by detection of P-glycoprotein (P-gp) measurement of P-gp-mediated drug transport and reversal of MDR by chemosensitizers. The in vitro chemosensitivity of three prostate cancer cell lines (PC-3, DU-145 and LNCaP) to doxorubicin was measured in a thymidine incorporation proliferation assay. Growth of the partially hormone-sensitive cell line LNCaP is inhibited by low doses of doxorubicin (IC50:27 ng./ml.), but PC-3 and DU-145 are highly resistant to the drug, with IC50 values of 10 micrograms./ml. and 7.5 micrograms./ml., respectively. The chemosensitivity of the PC-3 and DU-145 cells is increased in response to 1 microM. verapamil, 1 micrograms./ml. cyclosporine A and 2 microM. tamoxifen, which are known to partially reverse the MDR phenotype in other resistant tumors. A verapamil-sensitive drug efflux has been demonstrated for the PC-3 and Du-145, but not for the LNCaP, cell lines, using flow cytometric measurements of the P-gp substrate rhodamine 123 efflux from preloaded cells. In agreement with the functional measurements, the expression of the P-glycoprotein was detected in the PC-3 and Du-145 cell lines in Western blots using the monoclonal C 219 antibody. In conclusion, the chemoresistant and hormone-insensitive PC-3 and Du-145 cell lines express P-gp and exhibit verapamil-sensitive drug efflux, indicative of MDR. However, the low MDR-reversal rates observed in these cell lines in response to chemosensitizers in clinically achievable concentrations (approximately 2- to 3-fold reversal), point to non-MDR-associated cellular mechanisms as dominant factors of chemoresistance in prostate cancer.
The therapeutic responsiveness of genetically defined tumors expressing or devoid of the p53 tumor suppressor gene was compared in immunocompromised mice. Tumors expressing the p53 gene contained a high proportion of apoptotic cells and typically regressed after treatment with gamma radiation or adriamycin. In contrast, p53-deficient tumors treated with the same regimens continued to enlarge and contained few apoptotic cells. Acquired mutations in p53 were associated with both treatment resistance and relapse in p53-expressing tumors. These results establish that defects in apoptosis, here caused by the inactivation of p53, can produce treatment-resistant tumors and suggest that p53 status may be an important determinant of tumor response to therapy.
We have recently developed a micropuncture technique to assess repeatedly function of the same nephrons in chronic renal disease and subsequently examine the morphology of their glomeruli by serial thin-section histological analysis. Using this approach, a potential causal linkage between early functional patterns and late structural abnormalities was examined in glomeruli of two established rat models of glomerular sclerosis. The models are (a) puromycin aminonucleoside (PAN) administration in unilaterally nephrectomized Munich-Wistar rats and (b) adriamycin (ADM) treatment in nonnephrectomized Munich-Wistar rats. Single nephron GFR (SNGFR) and glomerular capillary hydraulic pressure (PGC) were measured repeatedly for 8 (PAN rats) or 31 wk (ADM rats). In all animals studied, values for PGC remained at, or slightly below, levels measured before PAN or ADM administration. SNGFR values declined progressively in all glomeruli in PAN rats. Although some glomeruli in ADM rats had an increase in SNGFR above levels observed in nonnephrectomized control rats, these hyperfiltering glomeruli did not have abnormally high PGC nor did they exhibit glomerular sclerosis at the completion of the study. Histological analysis revealed the existence of a significant inverse correlation between the degree of sclerosis and SNGFR assessed at the time of sacrifice in both PAN and ADM groups. Chronic administration of captopril, an angiotensin I converting enzyme inhibitor, in PAN rats substantially attenuated development of glomerular sclerosis without affecting PGC in earlier stages. The observations in these models indicate that glomerular hyperfiltration and hypertension are not required for the development of glomerular sclerosis in renal diseases, and angiotensin I converting enzyme inhibitor can exert its protective effect independently of its effect on glomerular capillary pressure.