Dehydroleucodine inhibits tumor growth in a preclinical melanoma model by inducing cell cycle arrest, senescence and apoptosis.

Malignant melanoma represents the fastest growing public health risk of all cancer types worldwide. Several strategies and anti-cancer drugs have been used in an effort to improve treatments, but the development of resistance to anti-neoplastic drugs remains the major cause of chemotherapy failure in melanomas. Previously, we showed that the sesquiterpene lactone, dehydroleucodine (DhL), promotes the accumulation of DNA damage markers, such as H2AX and 53BP1, in human tumor cells. Also DhL was shown to trigger either cell senescence or apoptosis in a concentration-dependent manner in HeLa and MCF7 cells. Here, we evaluated the effects of DhL on B16F0 mouse melanoma cells in vitro and in a pre-clinical melanoma model. DhL inhibited the proliferation of B16F0 cells by inducing senescence or apoptosis in a concentration-dependent manner. Also, DhL reduced the expression of the cell cycle proteins cyclin D1 and B1 and the inhibitor of apoptosis protein, survivin. In melanomas generated by subcutaneous injection of B16F0 cells into C57/BL6 mice, the treatment with 20 mg DhL /Kg/day in preventive, simultaneous and therapeutic protocols reduced tumor volumes by 70%, 60% and 50%, respectively. DhL treatments reduced the number of proliferating, while increasing the number of senescent and apoptotic tumor cells. To estimate the long-term effects of DhL, a mathematical model was applied to fit experimental data. Extrapolation beyond experimental time points revealed that DhL administration following preventive and therapeutic protocols is predicted to be more effective than simultaneous treatments with DhL in restricting tumor growth.

Hyperthermia effects on Hsp27 and Hsp72 associations with mismatch repair (MMR) proteins and cisplatin toxicity in MMR-deficient/proficient colon cancer cell lines.

PURPOSE: Hyperthermia is used in combination with conventional anticancer agents to potentiate their cytotoxicity. One of its key events is the synthesis of heat shock proteins (HSPs), which are able to associate with components from DNA repair mechanisms. However, little is known about their relationship with the mismatch repair system (MMR). Our aim was to study the effects of hyperthermia on cisplatin (cPt) sensitivity and to determine whether MLH1 and MSH2 associate with Hsp27 and Hsp72 in MMR-deficient(-)/-proficient(+) cells. MATERIALS AND METHODS: HCT116+ch2 (MMR-) and HCT116+ch3 (MMR+) cell lines were exposed to cPt with or without previous hyperthermia (42 °C, 1 h). Clonogenic survival assays, MTT, confocal immunofluorescence, immunoprecipitation, immunoblotting and flow cytometry were performed. RESULTS: Hyperthermia increased the cPt resistance in MMR- cells 1.42-fold. Immunofluorescence revealed that after cPt, Hsp27 and Hsp72 translocated to the nucleus and colocalisation coefficients between these proteins with MLH1 and MSH2 increased in MMR+ cells. Immunoprecipitation confirmed the interactions between HSPs and MMR proteins in control and treated cells. Hyperthermia pretreatment induced cell cycle arrest, increased p73 expression and potentiated cPt sensitivity in MMR+ cells. CONCLUSIONS: This is the first report showing in a MMR-/+ cellular model that MLH1 and MSH2 are client proteins of Hsp27 and Hsp72. Our study suggests that p73 might participate in the cellular response to hyperthermia and cPt in a MMR-dependent manner. Further functional studies will confirm whether HSPs cooperate with the MMR system in cPt-induced DNA damage response or whether these protein interactions are only the result of their chaperone functions.

HER2 and ß-catenin protein location: importance in the prognosis of breast cancer patients and their correlation when breast cancer cells suffer stressful situations.

In human breast cancer, ß-catenin localization has been related with disease prognosis. Since HER2-positive patients are an important subgroup, and that in breast cancer cells a direct interaction of ß-catenin/HER2 has been reported, in the present study we have explored whether ß-catenin location is related with the disease survival. The study was performed in a tumor bank from patients (n = 140) that did not receive specific anti-HER2 therapy. The proteins were detected by immunohistochemistry in serial sections, 47 (33.5%) patients were HER2-positive with a long follow-up. HER2-positive patients that displayed ß-catenin at the plasma membrane (completely surrounding the tumour cells) showed a significant better disease-free survival and overall survival than the patients showing the protein on other locations. Then we explored the dynamics of the co-expression of ß-catenin and HER2 in human MCF-7 and SKBR3 cells exposed to different stressful situations. In untreated conditions MCF-7 and SKBR3 cells showed very different ß-catenin localization. In MCF-7 cells, cadmium administration caused a striking change in ß-catenin localization driving it from plasma membrane to cytoplasmic and perinuclear areas and HER2 showed a similar localization patterns. The changes induced by cadmium were compared with heat shock, H2O2 and tamoxifen treatments. In conclusion, this study shows the dynamical associations of HER2 and ß-catenin and their changes in subcellular localizations driven by stressful situations. In addition, we report for the first time the correlation between plasma membrane associated ß-catenin in HER2-positive breast cancer and survival outcome, and the importance of the protein localization in breast cancer samples.

Luteal expression of thyroid hormone receptors during gestation and postpartum in the rat.

BACKGROUND: Progesterone (P4) is the main steroid secreted by the corpora lutea (CL) and is required for successful implantation and maintenance of pregnancy. Although adequate circulating levels of thyroid hormone (TH) are needed to support formation and maintenance of CL during pregnancy, TH signaling had not been described in this gland. We determined luteal thyroid hormone receptor isoforms (TR) expression and regulation throughout pregnancy and under the influence of thyroid status, and in vitro effects of triiodothyronine (T3) exposure on luteal P4 synthesis. METHODS: Euthyroid female Wistar rats were sacrificed by decapitation on gestational day (G) 5, G10, G15, G19, or G21 of pregnancy or on day 2 postpartum (L2). Hyperthyroidism and hypothyroidism were induced in female Wistar rats by daily administration of thyroxine (T4; 0.25 mg/kg subcutaneously) or 6-propyl-2-thiouracil (PTU; 0.1 g/L in drinking water), respectively. Luteal TR expression of mRNA was determined using real-time reverse-transcription quantitative polymerase chain reaction, and of protein using Western blot and immunohistochemistry. Primary cultures of luteal cells and of luteinized granulosa cells were used to study in vitro effects of T3 on P4 synthesis. In addition, the effect of T3 on P4 synthesis under basal conditions and under stimulation with luteinizing hormone (LH), prolactin (PRL), and prostaglandin E2 (PGE2) was evaluated. RESULTS: TRα1, TRα2, and TRß1 mRNA were present in CL, increasing during the first half and decreasing during the second half of pregnancy. At the protein level, TRß1 was abundantly expressed during gestation reaching a peak at G19 and decreasing afterwards. TRα1 was barely expressed during early gestation, peaked at G19, and diminished thereafter. Expression of TRß1 and TRα1 at the protein and mRNA level were not influenced by thyroid status. T3 neither modified P4 secretion from CL of pregnancy nor its synthesis in luteinized granulosa cells in culture. CONCLUSIONS: This study confirms for the first time the presence of TR isoforms in the CL during pregnancy and postpartum, identifying this gland as a TH target during gestation. TR expression is modulated in this tissue in accordance with the regulation of P4 metabolism, and the abrupt peripartum changes suggest a role of TH during luteolysis. However, TH actions on the CL do not seem to be related to a direct regulation of P4 synthesis.

Prognostic implication of HSPA (HSP70) in breast cancer patients treated with neoadjuvant anthracycline-based chemotherapy.

Neoadjuvant chemotherapy is used in patients with locally advanced breast cancer to reduce tumor size before surgery. Unfortunately, resistance to chemotherapy may arise from a variety of mechanisms. Heat shock proteins (HSPs), which are highly expressed in mammary tumor cells, have been implicated in anticancer drug resistance. In spite of the widely described value of HSPs as molecular markers in cancer, their implications in breast tumors treated with anthracycline-based neoadjuvant chemotherapy has been poorly explored. In this study, we have evaluated, by immunohistochemistry, the expression of HSP27 (HSPB1) and HSP70 (HSPA) in serial biopsies from locally advanced breast cancer patients (n = 60) treated with doxorubicin (DOX)- or epirubicin (EPI)-based monochemotherapy. Serial biopsies were taken at days 1, 3, 7, and 21, and compared with prechemotherapy and surgical biopsies. After surgery, the patients received additional chemotherapy with cyclophosphamide, methotrexate, and 5-fluorouracil. High nuclear HSPB1 and HSPA expressions were found in invasive cells after DOX/EPI administration (P < 0.001), but the drug did not affect the cytoplasmic expression of the HSPs. Infiltrating lymphocytes showed high nuclear HSPA (P < 0.01) levels at postchemotherapy. No correlations were found between HSPs expression and the clinical and pathological response to neoadjuvant therapy. However, in postchemotherapy biopsies, high nuclear (>31 % of the cells) and cytoplasmic HSPA expressions (>11 % of the tumor cells) were associated with better DFS (P = 0.0348 and P = 0.0118, respectively). We conclude that HSPA expression may be a useful prognostic marker in breast cancer patients treated with neoadjuvant DOX/EPI chemotherapy indicating the need to change the administered drugs after surgery for overcoming drug resistance.

In MMTV-Her-2/neu transgenic mammary tumors the absence of caveolin-1-/- alters PTEN and NHERF1 but not ß-catenin expression.

In a recent study, we have shown that in mammary tumors from mice lacking the Cav-1 gene, there are alterations in specific heat shock proteins as well as in tumor development. With this in mind, we have now investigated other proteins in the same mammary mouse tumor model (Her-2/neu expressing mammary tumors from Cav-1 wild type and Cav-1 null mice), to further comprehend the complex tumor-stroma mechanisms involved in regulating stress responses during tumor development. In this tumor model the cancer cells always lacked of Cav-1, so the KO influenced the Cav-1 in the stroma. By immunohistochemistry, we have found a striking co-expression of ß-catenin and Her-2/neu in the tumor cells. The absence of Cav-1 in the tumor stroma had no effect on expression or localization of ß-catenin and Her-2/neu. Both proteins appeared co-localized at the cell surface during tumor development and progression. Since Her-2/neu activation induces MTA1, we next evaluated MTA1 in the mouse tumors. Although this protein was found in numerous nuclei, the absence of Cav-1 did not alter its expression level. In contrast, significantly more PTEN protein was noted in the tumors lacking Cav-1 in the stroma, with the protein localized mainly in the nuclei. P-Akt levels were relatively low in tumors from both Cav-1 WT and Cav-1 KO mice. There was also an increase in nuclear NHERF1 expression levels in the tumors arising from Cav-1 KO mice. The data obtained in the MMTV-neu model are consistent with a role for Cav-1 in adjacent breast cancer stromal cells in modulating the expression and localization of important proteins implicated in tumor cell behavior.

Hsp27 (HSPB1): a possible surrogate molecular marker for loss of heterozygosity (LOH) of chromosome 1p in oligodendrogliomas but not in astrocytomas.

In oligodendrogliomas, 1p loss of heterozygosity (LOH) is a predictor of good prognosis and treatment response. In contrast, in uveal melanomas, LOH of chromosome 3 has been linked to poor prognosis and downregulation of Hsp27. In the present study, we have analyzed the expression of heat-shock proteins (Hsps) to characterize subtypes of gliomas and their histopathologic features and to correlate with other molecular markers including LOH of 1p. Biopsies from patients with primary gliomas (n = 65) were analyzed by immunohistochemistry, chromogenic in situ hybridization and fluorescent in situ hybridization and methylation-specific PCR (MSP). Elevated Hsp27 and total Hsp70 expression levels were associated with high-grade astrocytomas (p = 0.0001 and p = 0.01, respectively). In grade III oligodendrogliomas, the Hsp27 levels were significantly higher (p = 0.03). Low O6-methylguanine-DNA methyltransferase (MGMT) expression was associated with grade II astrocytomas. Elevated ß-catenin expression was associated with grade III/IV astrocytomas (p = 0.003); p53 (+) tumors were more frequently found in grade III/IV astrocytomas (p = 0,001). LOH on 1p was associated with oligodendroglial tumours. In addition, a higher Hsp27 expression correlated with LOH of 1p (p = 0.017); this was also tested in two glioma cell lines. MSP was successful in only six samples. No significant correlations were found for the other markers. In conclusion, in oligodendroglial tumors, Hsp27 appeared as a surrogate marker of LOH of 1p which could also help to predict the disease prognosis. In gliomas, p53, Hsp27, Hsp70, MGMT, and ß-catenin correlated with histopathological characteristics, suggesting that these markers could predict the disease outcome and the response to treatments.

Effects of hyperthermia on Hsp27 (HSPB1), Hsp72 (HSPA1A) and DNA repair proteins hMLH1 and hMSH2 in human colorectal cancer hMLH1-deficient and hMLH1-proficient cell lines.

PURPOSE: The objective of the present study was to examine the consequences of a mild hyperthermia in human tumour cell lines deficient and proficient in the DNA mismatch repair system (MMR) to advance our understanding on the relationship between MMR and heat shock proteins (HSPs). MATERIALS AND METHODS: The human colon carcinoma cell lines HCT116 (parent cells), HCT116 + ch2 (MMR-deficient), and HCT116 + ch3 (MMR-proficient) were used. Cells were incubated at 41°C and 42°C for 1 h and then at 37°C for 4 and 24 h. The expression of Hsp27 and Hsp72 was evaluated by immunocytochemistry. Hsp27, Hsp72, hMLH1 and hMSH2 levels were assessed by western blotting in nuclear and cytoplasmic fractions. The alkaline comet assay was used to evaluate the DNA damage. RESULTS: The mild hyperthermia significantly increased the protein expression levels of Hsp27 and Hsp72 in all cell lines, which was higher in the cytoplasm and nucleus of HCT116 + ch3 cells. We also observed that heat induced translocation of hMLH1 and hMSH2 proteins from the nucleus to the cytoplasm in HCT116 + ch3 cells. The comet assay revealed that HCT116 parent cells were more resistant to heat-induced DNA damage. However, the MMR-proficient and deficient cell lines repaired the DNA damage at the same rate. CONCLUSIONS: The present study demonstrates that hyperthermia induced the nuclear accumulation of Hsp27 and Hsp72 and affected the subcellular localisation of hMLH1 and hMSH2 in HCT116 + ch3 cells. Our findings suggest that the MMR system is not a direct determining factor for the different heat shock response in HCT116 cells.

Absence of caveolin-1 alters heat shock protein expression in spontaneous mammary tumors driven by Her-2/neu expression.

In a previous study, we measured caveolin-1 protein levels, both in the normal breast and in breast cancer. The study revealed no association between caveolin-1 expression in the epithelial compartment and clinical disease outcome. However, high levels of caveolin-1 in the stromal tissue surrounding the tumor associated strongly with reduced metastasis and improved survival. Using an animal model, we found that the onset of mammary tumors driven by Her-2/neu expression was accelerated in mice lacking caveolin-1. We have analysed the heat shock protein (Hsp) response in the tumors of mice lacking caveolin-1. In all cases, the mammary tumors were estrogen and progesterone receptor negative, and the levels of Her-2/neu (evaluated by immunohistochemistry) were not different between the caveolin-1 +/+ (n = 8) and the caveolin-1 -/- (n = 7) tumors. However, a significant reduction in the extent of apoptosis was observed in mammary tumors from animals lacking caveolin-1. While Bcl-2, Bax, and survivin levels in the tumors were not different, the amount of HSPA (Hsp70) was almost double in the caveolin-1 -/- tumors. In contrast, HSPB1 (Hsp27/Hsp25) levels were significantly lower in the caveolin-1 -/- tumors. The mammary tumors from caveolin-1 null mice expressed more HSPC4 (gp96 or grp94), but HSPC1 (Hsp90), HSPA5 (grp78), HSPD1 (Hsp60), and CHOP were not altered. No significant changes in these proteins were found in the stroma surrounding these tumors. These results demonstrate that the disruption of the Cav-1 gene can cause alterations of specific Hsps as well as tumor development.

Expression of NHERF1 in colonic tumors induced by 1,2-dimethylhydrazine in rats is independent of plasma ovarian steroids.

In normal embryonic fibroblasts, the Na(+)/H(+) exchanger regulator factor 1 (NHERF1) stabilizes E-cadherin/ß-catenin binding and the lack of NHERF1 expression promotes cell transformation thus acting as a tumor suppressor gene. We here tested the hypothesis that NHERF1 could act as a tumor suppressor gene in colon cancer as a mediator of estrogens' protective actions in colon carcinogenesis. We studied the expression and localization of NHERF1 and ß-catenin by immunohistochemistry in colonic tumors induced by 1,2 dimethylhidrazine (DMH) in Sprague-Dawley rats. One group of the rats treated with the carcinogen was ovariectomized (OVX) in the middle of the tumor induction, simulating a human menopausal condition. We observed a protective role of estrogens in colon cancer, as non-ovariectomized rats (DMH) had a reduced tumor area compared with the ovariectomized group (DMH + OVX; mean ± SE) 28.98 ± 4.65 vs. 67.58 ± 8.69 (p < 0.00380). Despite the lack of plasma estrogen stimulation, we found abundant expression of NHERF1 in colon tumors from ovariectomized rats. NHERF1 was mainly localized in the cytoplasm of the adenocarcinoma cells and lost the apical localization previously reported in normal colon tissue. We also detected expression of NHERF1 by western blot in the SW48, CACO-2, and HT29 colon cancer cell lines. Non-estrogenic factors in plasma or the tumor microenvironment may regulate NHERF1 expression in transformed colon epithelial cells. Further studies are required to understand the regulation of NHERF1 expression in colon cancer tissue.

Estrogens regulate the expression of NHERF1 in normal colon during the reproductive cycle of Wistar rats.

In breast cancer cell lines, the Na(+)/H(+) exchanger regulator factor 1 (NHERF1) gene is regulated at the transcriptional level by estrogens, the protein expression levels correlate with the presence of estrogen receptors and the effect is blocked by anti-estrogens. However, there is limited information regarding the regulation of NHERF1 by estrogens in normal colon tissue. The NHERF1 protein has an important role in the maintenance of the intestine ultrastructure. NHERF1-deficient mice showed defects in the intestinal microvilli as well as molecular alterations in brush border membrane proteins. Here, we have studied the expression of NHERF1 in normal rat colon and uterus during the reproductive cycle of Wistar rats. We found that NHERF1 expression in rat colon during the estral cycle is modified by estrogen levels: higher expression of NHERF1 was observed during the proestrous and estrous stages and lower expression in diestrous 1 when estrogen levels decreased. In uterus, NHERF1 was expressed in the apical region of the luminal epithelium and glands in all stages of the estral cycle, and in both colon and uterus, the expression was independent of the proliferation status. Our results show that NHERF1 expression is regulated by estrogens in colon during the rat estral cycle.

P-cadherin and beta-catenin are useful prognostic markers in breast cancer patients; beta-catenin interacts with heat shock protein Hsp27.

The cadherin-catenin proteins have in common with heat shock proteins (HSP) the capacity to bind/interact proteins of other classes. Moreover, there are common molecular pathways that connect the HSP response and the cadherin-catenin protein system. In the present study, we have explored whether in breast cancer the HSP might interact functionally with the cadherin-catenin cell adhesion system. Beta-catenin was immunoprecipitated from breast cancer biopsy samples, and the protein complexes isolated in this way were probed with antibodies against HSP family members. We are thus the first to demonstrate a specific interaction between beta-catenin and Hsp27. However, beta-catenin did not bind Hsp60, Hsp70, Hsp90, gp96, or the endoplasmic reticulum stress response protein CHOP. To confirm the finding of Hsp27-beta-catenin interaction, the 27-kDa immunoprecipitated band was excised from one-dimensional polyacrylamide gel electrophoresis gels and submitted to liquid chromatography-tandem mass spectrometry with electrospray ionization, confirming a role for Hsp27. In addition, beta-catenin interacted with other proteins including heat shock transcription factor 1, P-cadherin, and caveolin-1. In human breast cancer biopsy samples, beta-catenin was coexpressed in the same tumor areas and in the same tumor cells that expressed Hsp27. However, this coexpression was strong when beta-catenin was present in the cytoplasm of the tumor cells and not when beta-catenin was expressed at the cell surface only. Furthermore, murine breast cancer cells transfected with hsp25 showed a redistribution of beta-catenin from the cell membrane to the cytoplasm. When the prognostic significance of cadherin-catenin expression was examined by immunohistochemistry in breast cancer patients (n = 215, follow-up = >10 years), we found that the disease-free survival and overall survival were significantly shorter for patients expressing P-cadherin and for patients showing expression of beta-catenin in the cytoplasm only (not at the cell surface). The interactions of beta-catenin with Hsp27 and with HSF1 may explain some of the molecular pathways that influence tumor cell survival and the clinical significance in the prognosis of the breast cancer patients.

Prognostic value of Bcl-2 in breast cancer patients treated with neoadjuvant anthracycline based chemotherapy.

We have analyzed the predictive/prognostic value of Bcl-2 protein in breast cancer patients treated with neoadjuvant chemotherapy. One hundred and ten patients were submitted to two different chemotherapeutic regimens: a) 5-fluorouracil, adriamycin or epirubicin, and cyclophosphamide (FAC/FEC) during 2-6 cycles before surgery and 3 or 4 additional cycles of FAC/FEC after surgery (n=40) and b) doxorubicin (D) 75 mg/m(2) or epirubicin (E) 120 mg/m(2) during 4 cycles before surgery, and 6 cycles of cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) after surgery (n=70). Bcl-2 expression, evaluated by immunohistochemistry, did not change significantly after chemotherapy and was not related to clinical/pathological response. In FAC/FEC group, Bcl-2 positive expression after chemotherapy correlated with better disease free survival (DFS) and overall survival (OS) (P=0.008 and P=0.001). In D/E group, Bcl-2 also correlated with better DFS and OS (P=0.03 and P=0.054) in the post-chemotherapy biopsies. An unusual nuclear localization of Bax was observed in some biopsies, but this localization did not correlate with the tumor response or outcome of the patients. We found that a high Bcl-2 expression had no predictive value but had prognostic value in breast cancer patients treated with neoadjuvant anthracycline based chemotherapy.

A pilot study with a therapeutic vaccine based on hydroxyapatite ceramic particles and self-antigens in cancer patients.

We describe an approach to produce an autologous therapeutic antitumor vaccine using hydroxyapatite (HA) for vaccinating cancer patients. The novel approach involved (1) the purification of part of the self-tumor antigens/ adjuvants using column chromatography with HA, (2) the employ of HA as a medium to attract antigen-presenting cells (APCs) to the vaccination site, and (3) the use of HA as a vector to present in vivo the tumor antigens and adjuvants to the patient's APCs. The vaccine was prepared using and combining HA particles, with at least 3 heat shock proteins (gp96 was one of them possibly with chaperoned proteins/peptides as shown in the slot blots) and with proteins from the cell membrane system (including Hsp70, Hsp27, and membrane proteins). The timing of HA degradation was tested in rats; the HA particles administered under the skin attracted macrophages and were degraded into smaller particles, and they were totally phagocytized within 1 week. In patients (n = 20), the vaccine was then administered weekly and showed very low toxicity, causing minor and tolerable local inflammation (erythema, papule, or local pain); only 1 patient who received a larger dose presented hot flashes, and there were no systemic manifestations of toxicity or autoimmune diseases attributed to the vaccine. Our study suggests that this therapeutic vaccine has shown some efficacy producing a positive response in certain patients. Stable disease was noted in 25% of the patients (renal carcinoma, breast carcinoma, and astrocytoma), and a partial response was noted in 15% of the patients (breast carcinoma and astrocytoma). The most encouraging results were seen in patients with recurrent disease; 4 patients in these conditions (20%) are disease free following the vaccine administration. However, we do not want to overstate the clinical efficacy in this small number of patients. The therapeutic vaccine tested in our study is working by activating the T-cell response as was shown in the comparative histological and immunohistochemical study performed in the pre- and postvaccine biopsy taken from a patient with inflammatory breast carcinoma. However, we cannot ruled out that the vaccine could also be producing an antibody(ies)-mediated response. In conclusion, this therapeutic vaccine based on HA ceramic particles and self-antigens can be safely administered and is showing some encouraging clinical results in cancer patients.

11,13-dihydro-dehydroleucodine, a derivative of dehydroleucodine with an inactivated alkylating function conserves the anti-proliferative activity in G2 but does not cause cytotoxicity.

Modulation of vascular smooth muscle cell (VSMC) proliferation has critical therapeutic implications for vascular disease. Recently, we demonstrated that the sesquiterpene lactone dehydroleucodine (DhL) inhibited the proliferation of VSMCs in G2 phase. It is known that the alpha,beta-unsaturated carbonyl group of the sesquiterpene lactone has a nonspecific alkylating activity that inhibits a large number of enzymes or factors involved in key biological processes. We analyzed whether the DhL alpha-methylene-gamma-lactone function is directly involved in cell proliferation arrest in G2 and in cell toxicity. To this end, the effects of both DhL and 11,13-dihydro-dehydroleucodine (2H-DhL), a derivative of DhL with inactivated alpha-methylenelactone function, on cultured VSMC viability and proliferation were assessed. We found that both DhL and 2H-DhL inhibited the proliferation of VSMCs in a dose-dependent manner, inducing a transient arrest in G2 phase. DhL, but not 2H-DhL, had a cytotoxic effect at concentrations up to 12 microM, indicating that cell proliferation arrest and cytotoxicity are mediated by different cellular targets. From these results we infer that only 2H-DhL is able to arrest cell proliferation in G2 without affecting cell viability at any concentration.

Hsp25 and Hsp70 in rodent tumors treated with doxorubicin and lovastatin.

Heat shock protein 27 (Hsp27) and Hsp70 have been involved in resistance to anticancer drugs in human breast cancer cells growing in vitro and in vivo. In this study, we examined the expression of Hsp25 (the rodent homologue to human Hsp27) and Hsp70 in 3 different rodent tumors (a mouse breast carcinoma, a rat sarcoma, and a rat lymphoma maintained by subcutaneous passages) treated in vivo with doxorubicin (DOX) and lovastatin (LOV). All tumors showed massive cell death under control untreated conditions, and this massive death increased after cytotoxic drug administration. In this study, we show that this death was due to classic apoptosis. The tumors also showed isolated apoptotic cells between viable tumor cells, and this occurred more significantly in the lymphoma. The tumor type that was more resistant to cell death was the sarcoma, and this was found in sarcomas growing both under control conditions and after cytotoxic drug administration. Moreover, sarcomas showed the highest expression levels of Hsp25 in the viable tumor cells growing under untreated conditions, and these levels increased after DOX and LOV administration. After drug treatment, only sarcoma tumor cells showed a significant increase in Hsp70. In other words, sarcomas were the tumors with lower cell death, displayed a competent Hsp70 and Hsp25 response with nuclear translocation, and had the highest levels of Hsp25. In sarcomas, Hsp25 and Hsp70 were found in viable tumor cells located around the blood vessels, and these areas showed the most resistant tumor cell phenotype after chemotherapy. In addition, Hsp25 expression was found in endothelial cells as unique feature revealed only in lymphomas. In conclusion, our study shows that each tumor type has unique features regarding the expression of Hsp25 and Hsp70 and that these proteins seem to be implicated in drug resistance mainly in sarcomas, making these model systems important to perform more mechanistic studies on the role of Hsps in resistance to certain cytotoxic drugs.

Heat shock proteins HSP27 and HSP70 in unilateral obstructed kidneys.

Unilateral ureteropelvic junction obstruction (UPJO), a condition involving injury to the kidney, leads to vasoconstriction, macrophage infiltration, oxidative stress, and tubulointerstitial fibrosis. In the present study we analyzed in 22 children, the impact of UPJO on expression of HSP27 and HSP70 and related the changes to renal function and duration of obstruction. Kidney function was evaluated, and renal biopsies were obtained for immunohistochemical and western blot analyses at the time of surgery. Kidney tissue from 5 children of similar age, removed because of various malignancies, was used as control. Increased HSP27, with an expression pattern related to the duration of the obstruction, was noted in proximal tubules (PTs), cortical collecting ducts (CCDs), and medullary collecting ducts. Strong HSP27 staining in cytoplasm and nuclei of these segments appeared in children with kidney obstruction for 2.1+/-0.41 years. HSP70 showed a close spatial expression pattern with that of HSP27. The increased staining of HSP70 in PTs and CCDs was related to the decreased glomerular filtration rate. The constitutive/cognate form of HSP70 (HSC72) was absent. These results support the concept that HSP27 and HSP70 are involved in the adaptive response of the human kidney to congenital UPJO.

Deoxyribonucleic acid damage induced by doxorubicin in peripheral blood mononuclear cells: possible roles for the stress response and the deoxyribonucleic acid repair process.

Doxorubicin is an antineoplastic drug widely used in cancer treatment. However, many tumors are intrinsically resistant to the drug or show drug resistance after an initial period of response. Among the different molecules implicated with doxorubicin resistance are the heat shock proteins (Hsps). At present we do not know with certainty the mechanism(s) involved in such resistance. In the present study, to advance our knowledge on the relationship between Hsps and drug resistance, we have used peripheral blood mononuclear cells obtained from healthy nonsmoker donors to evaluate the capacity of a preliminary heat shock to elicit the Hsp response and to establish the protection against the deoxyribonucleic acid (DNA) damage induced by doxorubicin. DNA damage and repair were determined using the alkaline comet assay. We also measured the expression of Hsp27, Hsp60, Hsp70, Hsp90, hMLH1, hMSH2, and proliferating cell nuclear antigen by immunocytochemistry. The damage induced by doxorubicin was more efficiently repaired when the cells were previously heat shocked followed by a resting period of 24 hours before drug exposure, as shown by (1) the increased number of undamaged cells (P < 0.05), (2) the increased DNA repair capacity (P < 0.05), and (3) the high expression of the mismatch repair (MMR) proteins hMLH1 and hMSH2 (P < 0.05). In addition, in the mentioned group of cells, we confirmed by Western blot high expression levels of Hsp27 and Hsp70. We also noted a nuclear translocation of Hsp27 and mainly of Hsp70. Furthermore, inducible Hsp70 was more expressed in the nucleus than Hsc70, showing a possible participation of Hsp70 in the DNA repair process mediated by the MMR system.