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

Serological detection of heat shock protein hsp27 in normal and breast cancer patients

Heat shock protein Mr 27,000 (hsp27) is found in many human breast cancer cells and tissues; its expression is associated with the presence of estrogen receptors, lower cell proliferation, and resistance to certain chemotherapies. The purpose of this study was to assess whether hsp27 may be present in sera from women with primary breast cancer and to know whether autoantibodies to hsp27 may be found in these patients. The study was performed by Western blot analyzing sera from 42 normal premenopausal women, 20 normal postmenopausal women, and 36 breast cancer patients. hsp27 was clearly detected in sera by immunoblotting but only after immunoprecipitation. The mean hsp27 levels in cancer patients were higher than in the control patients; however, 66% of the breast cancer patients showed hsp27 within the normal range, indicating low sensitivity. Moreover, cancer patients with metastatic disease did not show significantly higher hsp27 levels than cancer patients without metastases. Serum hsp27 levels did not correlate with the hsp27 levels in tumor tissues detected by immunohistochemistry. Elevated CA 15-3 levels were not associated with high hsp27 values. Autoantibodies against hsp27 were not detected by immunoblotting in normal sera and in sera from breast cancer patients. As a consequence, serological determination of this biomarker is unlikely to be of utility in the detection and follow-up of breast cancer patients

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

The sesquiterpene lactone dehydroleucodine triggers senescence and apoptosis in association with accumulation of DNA damage markers.

Sesquiterpene lactones (SLs) are plant-derived compounds that display anti-cancer effects. Some SLs derivatives have a marked killing effect on cancer cells and have therefore reached clinical trials. Little is known regarding the mechanism of action of SLs. We studied the responses of human cancer cells exposed to various concentrations of dehydroleucodine (DhL), a SL of the guaianolide group isolated and purified from Artemisia douglasiana (Besser), a medicinal herb that is commonly used in Argentina. We demonstrate for the first time that treatment of cancer cells with DhL, promotes the accumulation of DNA damage markers such as phosphorylation of ATM and focal organization of γH2AX and 53BP1. This accumulation triggers cell senescence or apoptosis depending on the concentration of the DhL delivered to cells. Transient DhL treatment also induces marked accumulation of senescent cells. Our findings help elucidate the mechanism whereby DhL triggers cell cycle arrest and cell death and provide a basis for further exploration of the effects of DhL in in vivo cancer treatment models

DhL treatment inhibits cell proliferation in a dose-dependent manner.

<p>Unsynchronized HeLa (A) and MCF-7 cells (B) and synchronized HeLa (C and D) cells were treated with 0, 5, 10, 20, or 30 µM DhL for 72 or 96 h and counted every 24 h. The total number of cells counted each 24 h (Total cells) were compared with the number of viable cells (Viable cells) (D). Data are expressed as the mean ± SEM of 3 independent experiments. (A), (B) and (C) * p≤0.05, ** p≤0.01, *** p≤0.001 vs. control group (0 µM DhL). (D) * p≤0.05, ** p≤0.01, *** p≤0.001 total cells vs. viable cells.</p

DhL induces cellular senescence in HeLa cells.

<p>Synchronized HeLa cells treated with 0–20 µM DhL for 48 h were analyzed for: (A) protein concentration and (B) SA-β-Gal activity (at pH 6) in cell extracts. Equal volumes of supernatants were assayed. C) SA-β-Gal activity at pH 6 <i>in situ</i>. Representative panels for control and treated cells stained for SA-β-Gal and examined by bright field microscopy are shown. Bar: 50 µm. Right: percentages of SA-β-Gal-positive cells. (D) Senescence-associated heterochromatin foci (SAHF) in control and treated cells stained with DAPI and examined by bright field and fluorescence microscopy (merge: bright field/DAPI) and fluorescence microscopy (DAPI). The insets are magnifications of the boxed areas in the DAPI column. Arrowheads indicate the SAHF. Bar: 10 µm. Right: percentages of SAHF-positive cells. (E) Synchronized HeLa cells treated with 20 µM DhL and then with fresh medium plus DMSO (“Fresh medium”) for the time indicated in the upper panel (treatment 1–4) or with fresh medium for 24 h (treatment 5) were analyzed for protein concentration and for SA-β-Gal activity at pH 6. The protein concentration (lower left) and SA-β-Gal activity (lower right) in cell extracts from each treatment are shown. Equal volumes of extract were assayed. Data represents the mean ± SEM of 2 experiments. * p≤0.05, ** p≤0.01, *** p≤0.001 vs. control group (0 µM).</p

Treatment with 30 µM DhL induces apoptosis in HeLa cells.

<p>Unsynchronized (A) and synchronized (B) HeLa cells were treated with 0, 20, or 30 µM DhL for 24 or 48 h. DNA content was assessed by flow cytometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053168#pone.0053168.s002" target="_blank">Fig. S2</a>, DNA distributions). The percentages of unsynchronized and synchronized cells in the sub-G1 phase are shown. (C) Synchronized HeLa cells were treated with 0, 20, or 30 µM DhL for 24 or 48 h. Apoptotic cells were assessed by TUNEL assay. Left: representative panels with apoptotic cells indicated by arrowheads. Bar: 50 µm. Right: percentages of apoptotic cells at 24 and 48 h. (D) Unsynchronized HeLa cells were treated as in (C) and subjected to Annexin V assay. Left: representative panels with apoptotic cells stained with Annexin V (bright cells) at 24 h. Representative fields of Annexin V positive cells for 48 and 72 h treatment are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053168#pone.0053168.s002" target="_blank">Fig. S2</a> C. Bar: 50 µm. Right: percentages of apoptotic cells at 24, 48 and 72 h. Data represent mean ± SEM of 3 independent experiments. * p≤0.05, ** p≤0.01, *** p≤0.001 vs. control group (0 µM DhL).</p