The nitroxide Tempol modulates anthracycline resistance in breast cancer cells

The occurrence of multidrug resistance (MDR) is the major obstacle to successful anthracycline-based cancer chemotherapy. In the present study, we assessed the effects of Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, TPL), a piperidine nitroxide with growth-inhibitory properties in tumor cell lines, on a number of molecular mechanisms involved in the resistance of human breast adenocarcinoma cell lines to doxorubicin (DOX). Cytotoxicity studies in MCF-7 wildtype and their MDR variant MCF-7 Adr(R) cells showed a synergistic effect between TPL and DOX when exposure to TPL preceded or was simultaneous with DOX treatment in MCF-7 Adr(R) cells. This effect of TPL seems to be due in part to its ability to increase peroxide levels and to deplete cellular glutathione pools. In addition, TPL increased DOX accumulation in MCF-7 Adr(R) cells by interfering with P-glycoprotein-mediated DOX efflux, as evidenced using a specific antibody that recognizes the active form of the protein. TPL was also found to affect the expression levels of proteins involved in response to drug treatment (e.g., p53, bcl2, bax, p21). Taken together, our results indicate that TPL is a potential new agent that may improve the clinical effect of DOX in tumors exhibiting a MDR phenotype.     

Synthesis and evaluation of nitric oxide-releasing DDB derivatives as potential Pgp-mediated MDR reversal agents in MCF-7/Adr cells

Novel furoxan-based nitric oxide (NO)-releasing DDB derivatives (7a-j) were synthesized. Compounds 7i and 7j significantly reversed the resistance of MCF-7/Adr cells to doxorubicin in the combination treatment, and markedly increased the intracellular accumulation of doxorubicin probably via inhibiting Pgp-mediated intracellular drug efflux as well as down-regulating doxorubicin-induced Pgp expression. It was demonstrated that NO released by 7i and 7j played an important role in increasing intracellular doxorubicin accumulation and chemo-sensitizing MCF-7/Adr cells to doxorubicin, and the synergic effects of DDB and NO-donor moieties in 7i and 7j may contribute to reversing Pgp-mediated MDR in MCF-7/Adr cells to doxorubicin.     

Reversal of doxorubicin resistance by guggulsterone of Commiphora mukul in vivo

Our previous study has shown co-administration of guggulsterone resulted in significant increase in chemosensitivity of multidrug-resistant human breast cancer MCF-7/DOX cells to doxorubicin (DOX) in vitro. The present study was designed to investigate whether guggulsterone had the similar modulatory activities in vivo. MCF-7/DOX and MCF-7 xenograft mice models were established. At the end of the experiment (day 28), doxorubicin treatment alone did not significantly inhibit tumor growth in MCF-7/DOX xenograft, indicating that it retained doxorubicin resistance. Whereas, doxorubicin treatment alone significantly inhibited tumor growth in MCF-7 xenograft, suggesting that it maintained doxorubicin sensitivity. When doxorubicin and guggulsterone were co-administrated, their antitumor activities were augmented in MCF-7/DOX xenograft. However, combination therapy did not enhance the antitumor effects of doxorubicin in MCF-7 xenograft. The expression of proliferative cell nuclear antigens PCNA and Ki67 after doxorubicin treatment alone was not significantly different from that of vehicle group in MCF-7/DOX xenograft. On the contrary, doxorubicin treatment alone significantly reduced PCNA and Ki67 expression in MCF-7 xenograft. Combination therapy also significantly reduced PCNA and Ki67 expression in MCF-7/DOX xenograft, compared to doxorubicin treatment alone. However, combination therapy did not enhance the inhibitory effects of doxorubicin on PCNA and Ki67 expression in MCF-7 xenograft. Examining the apoptotic index by TUNEL assay showed similar results. Further studies demonstrated the inhibitory effects of guggulsterone on Bcl-2 and P-glycoprotein expression were the possible reason to increase chemosensitivity of MCF-7/DOX cells to doxorubicin in vivo. Examining body weight, hematological parameters, hepatic, cardiac and gastrointestinal tracts histopathology revealed that no significant signs of toxicity were related to guggulsterone. Guggulsterone might reverse doxorubicin resistance in vivo, with no severe side effects.     

Ascorbate promotes the cellular accumulation of doxorubicin and reverses the multidrug resistance in breast cancer cells by inducing ROS-dependent ATP depletion

Chemotherapy is the most effective strategy for the treatment of metastatic breast cancer. However, P-glycoprotein (P-gp)-mediated multidrug resistance severely limits the efficacy of chemotherapy and is a major cause of the failure during chemotherapeutic treatment. In this study, we investigated the anticancer effects of combining chemotherapeutic drugs with ascorbate (AA) in human breast cancer cells. We found that combined administration of AA can improve the sensitivity of both MCF-7 and doxorubicin (Dox)-resistant MCF-7/Adr cells to Dox in vitro and in vivo by a reactive oxygen species (ROS)-dependent mechanism. Further studies proved that AA can promote the accumulation of Dox in MCF-7/Adr cells when combined with doxorubicin. AA had no effect on the expression of P-gp at the mRNA and protein levels, but could decrease its activity as demonstrated by an obvious inhibition of efflux of P-gp substrate Rh 123. AA reduced ATP levels in both MCF-7 and MCF-7/Adr cells, and pretreating AA-stimulating cells with catalase completely rescued ATP levels. With ATP reduction, we observed an increased cellular calcium and the appearance of vacuoles and micropores on the cell surface, indicating the increased cell membrane permeability in AA-treated MCF-7/Adr cells. The above results suggest that AA could promote the cellular accumulation of doxorubicin by inducing ROS-dependent ATP depletion. Clinically, a combination of AA with doxorubicin would be a novel strategy for reversal of the multidrug resistance in human breast cancer cells during chemotherapy.     

Down-regulation of cellular FLICE-inhibitory protein (Long Form) contributes to apoptosis induced by Hsp90 inhibition in human lung cancer cells

Background

Breast cancer is the most common cancer in the Arab world and it ranked first among Saudi females. Doxorubicin (DOX), an anthracycline antibiotic is one of the most effective anticancer agents used to treat breast cancer. chronic cardiotoxicity is a major limiting factor of the use of doxorubicin. Therefore, our study was designed to assess the role of a natural product resveratrol (RSVL) on sensitization of human breast cancer cells (MCF-7) to the action of DOX in an attempt to minimize doxorubicin effective dose and thereby its side effects.

Methods

Human breast cancer cell line MCF-7, was used in this study. Cytotoxic activity of DOX was determined using (sulforhodamine) SRB method. Apoptotic cells were quantified after treatment by annexin V-FITC- propidium iodide (PI) double staining using flow-cytometer. Cell cycle disturbance and doxorubicin uptake were determined after RSVL or DOX treatment.

Results

Treatment of MCF-7 cells with 15 μg/ml RSVL either simultaneously or 24 h before DOX increased the cytotoxicity of DOX, with IC50 were 0.056 and 0.035 μg/ml, respectively compared to DOX alone IC50 (0.417 μg/ml). Moreover, flow cytometric analysis of the MCF-7 cells treated simultaneously with DOX (0.5 μg/ml) and RSVL showed enhanced arrest of the cells in G₀ (80%). On the other hand, when RSVL is given 24 h before DOX although there was more increased in the cytotoxic effect of DOX against the growth of the cells, however, there was decreased in percentage arrest of cells in G₀, less inhibition of DOX-induced apoptosis and reduced DOX cellular uptake into the cells.

Conclusion

RSVL treatment increased the cytotoxic activity of DOX against the growth of human breast cancer cells when given either simultaneously or 24 h before DOX.     

Chemical ionization mass spectrometric determination of acrolein in human breast cancer cells

A selected ion flow tube-chemical ionization mass spectrometric method is presented for the first determination of acrolein metabolically produced in biological tissues. Acrolein in aqueous samples (2.5 ml) is preconcentrated by distillation and directly analyzed using gas-phase proton transfer from H3O+. This method provides sensitive detection of acrolein with the method detection limit of 15 nM at the 99% confidence level. Detection is linear up to the highest concentration studied (13.5 microM, R2 = 0.998). Acrolein levels are determined in doxorubicin-sensitive (MCF-7) and doxorubicin-resistant (MCF-7/Adr) human breast cancer cells in vitro. The intracellular acrolein concentrations differ insignificantly: 0.61 microM for sensitive cells and 0.54 microM for resistant cells. Treatment with a physiological concentration of doxorubicin (0.5 microM) for 24 h at 37 degrees C increased acrolein levels by factors of 2.6 and 1.9 for MCF-7 and MCF-7/Adr cells, respectively. The differential enhancement observed is consistent with the lower levels of enzymes that neutralize oxidative stress in sensitive MCF-7 cells and overexpression of an active drug efflux pump P-170 glycoprotein in resistant MCF-7/Adr cells.     

Hsa_circ_0092276 promotes doxorubicin resistance in breast cancer cells by regulating autophagy via miR-348/ATG7 axis

Previous study has confirmed that hsa_circ_0092276 is highly expressed in doxorubicin (DOX)-resistant breast cancer cells, indicating that hsa_circ_0092276 may be involved in regulating the chemotherapy resistance of breast cancer. Here we attempted to investigate the biological role of hsa_circ_0092276 in breast cancer. We first constructed DOX-resistant breast cancer cells (MCF-7/DOX and MDA-MB-468/DOX). The 50% inhibiting concentration of MCF-7/DOX and MDA-MB-468/DOX cells was significantly higher than that of their parental breast cancer cells, MCF-7 and MDA-MB-46. MCF-7/DOX and MDA-MB-468/DOX cells also exhibited an up-regulation of drug resistance-related protein MDR1. Compared with MCF-7 and MDA-MB-46 cells, hsa_circ_0092276 was highly expressed in MCF-7/DOX and MDA-MB-468/DOX cells. Hsa_circ_0092276 overexpression enhanced proliferation and the expression of LC3-II/LC3-I and Beclin-1, and repressed apoptosis of breast cancer cells. The effect of hsa_circ_0092276 up-regulation on breast cancer cells was abolished by 3-methyladenine (autophagy inhibitor). Hsa_circ_0092276 modulated autophagy-related gene 7 (ATG7) expression via sponging miR-384. Hsa_circ_0092276 up-regulation promoted autophagy and proliferation, and repressed apoptosis of breast cancer cells, which was abolished by miR-384 overexpression or ATG7 knockdown. In addition, LV-circ_0092276 transfected MCF-7 cell transplantation promoted autophagy and tumor growth of breast cancer in mice. In conclusion, our data demonstrate that hsa_circ_0092276 promotes autophagy and DOX resistance in breast cancer by regulating miR-348/ATG7 axis. Thus, this article highlights a novel competing endogenous RNA circuitry involved in DOX resistance in breast cancer.     

Comparative study of p53 expression in human carcinoma cell lines A549 and MCF7 under anticancer drug treatment

The effect of anticancer drugs on the expression of p53 protein in tumor cells was studied using the Western Blot analysis. Human lung carcinoma cell line A549 and human breast carcinoma cell line MCF7 sensitive (WT) and resistant (DOX/R) to doxorubicin were used. An increase in p53 protein expression was found in A549 and MCF7 (WT) cells treated with cisplatin, methotrexate, and doxorubicin, whereas the level of p53 was not statistically significantly changed in the MCF7 DOX/R cells. In the untreated MCF7 DOX/R cells the level of p53 protein was markedly higher than in the untreated WT MCF7 cells. A potential role of p53 protein in the development of doxorubicin-resistance in carcinoma cells is discussed.     

Systematic expression analysis of genes related to multidrug-resistance in isogenic docetaxel- and adriamycin-resistant breast cancer cell lines

Docetaxel (Doc) and adriamycin (Adr) are two of the most effective chemotherapeutic agents in the treatment of breast cancer. However, their efficacy is often limited by the emergence of multidrug resistance (MDR). The purpose of this study was to investigate MDR mechanisms through analyzing systematically the expression changes of genes related to MDR in the induction process of isogenic drug resistant MCF-7 cell lines. Isogenic resistant sublines selected at 100 and 200 nM Doc (MCF-7/100 nM Doc and MCF-7/200 nM Doc) or at 500 and 1,500 nM Adr (MCF-7/500 nM Adr and MCF-7/1,500 nM) were developed from human breast cancer parental cell line MCF-7, by exposing MCF-7 to gradually increasing concentrations of Doc or Adr in vitro. Cell growth curve, flow cytometry and MTT cytotoxicity assay were preformed to evaluate the MDR characteristics developed in the sublines. Some key genes on the pathways related to drug resistance (including drug-transporters: MDR1, MRP1 and BCRP; drug metabolizing-enzymes: CYP3A4 and glutathione S-transferases (GST) pi; target genes: topoisomerase II (TopoIIα) and Tubb3; apoptosis genes: Bcl-2 and Bax) were analyzed at RNA and protein expression levels by real time RT-qPCR and western blot, respectively. Compared to MCF-7/S (30.6 h), cell doubling time of MCF-7/Doc (41.6 h) and MCF-7/Adr (33.8 h) were both prolonged, and the cell proportion of resistant sublines in G1/G2 phase increased while that in S-phase decreased. MCF-7/100 nM Doc and MCF-7/200 nM Doc was 22- and 37-fold resistant to Doc, 18- and 32-fold to Adr, respectively. MCF-7/500 nM Adr and MCF-7/1,500 nM Adr was 61- and 274-fold resistant to Adr, three and 12-fold to Doc, respectively. Meantime, they also showed cross-resistance to the other anticancer drugs in different degrees. Compared to MCF-7/S, RT-qPCR and Western blot results revealed that the expression of MDR1, MRP1, BCRP, Tubb3 and Bcl-2 were elevated in both MCF-7/Doc and MCF-7/Adr, and TopoIIα, Bax were down-regulated in both the sublines, while CYP3A4, GST pi were increased only in MCF-7/Doc and MCF-7/Adr respectively. Furthermore, the changes above were dose-dependent. The established MCF-7/Doc or MCF-7/Adr has the typical MDR characteristics, which can be used as the models for resistance mechanism study. The acquired process of MCF-7/S resistance to Doc or Adr is gradual, and is complicated with the various pathways involved in. There are some common resistant mechanisms as well as own drug-specific changes between both the sublines.     

Small-molecule synthetic compound norcantharidin reverses multi-drug resistance by regulating Sonic hedgehog signaling in human breast cancer cells

Multi-drug resistance (MDR), an unfavorable factor compromising treatment efficacy of anticancer drugs, involves upregulated ATP binding cassette (ABC) transporters and activated Sonic hedgehog (Shh) signaling. By preparing human breast cancer MCF-7 cells resistant to doxorubicin (DOX), we examined the effect and mechanism of norcantharidin (NCTD), a small-molecule synthetic compound, on reversing multidrug resistance. The DOX-prepared MCF-7R cells also possessed resistance to vinorelbine, characteristic of MDR. At suboptimal concentration, NCTD significantly inhibited the viability of DOX-sensitive (MCF-7S) and DOX-resistant (MCF-7R) cells and reversed the resistance to DOX and vinorelbine. NCTD increased the intracellular accumulation of DOX in MCF-7R cells and suppressed the upregulated the mdr-1 mRNA, P-gp and BCRP protein expression, but not the MRP-1. The role of P-gp was strengthened by partial reversal of the DOX and vinorelbine resistance by cyclosporine A. NCTD treatment suppressed the upregulation of Shh expression and nuclear translocation of Gli-1, a hallmark of Shh signaling activation in the resistant clone. Furthermore, the Shh ligand upregulated the expression of P-gp and attenuated the growth inhibitory effect of NCTD. The knockdown of mdr-1 mRNA had not altered the expression of Shh and Smoothened in both MCF-7S and MCF-7R cells. This indicates that the role of Shh signaling in MDR might be upstream to mdr-1/P-gp, and similar effect was shown in breast cancer MDA-MB-231 and BT-474 cells. This study demonstrated that NCTD may overcome multidrug resistance through inhibiting Shh signaling and expression of its downstream mdr-1/P-gp expression in human breast cancer cells.     

TNF-alpha promotes Doxorubicin-induced cell apoptosis and anti-cancer effect through downregulation of p21 in p53-deficient tumor cells

p53 is a key regulator in cell apoptosis, and cancer cells deficient in p53 expression fail to respond to chemotherapy. Here we show that effective Doxorubicin (DOX)-induced apoptosis is p53-dependent. However, an alternative treatment of DOX/TNF-alpha/DOX restored sensitivity of p53-deficient cells to DOX-induced apoptosis. Treatment of cells with TNF-alpha resulted in a decrease of p21 (waf1/cip1/sdi1) expression following second dose of DOX. In previous work, we demonstrated that p21 suppressed DOX-induced apoptosis via its (cyclin-dependent kinase) CDK-binding and CDK-inhibitory activity. Thus, we propose that TNF-alpha enhances the anti-cancer effect of DOX through suppressing the anti-apoptotic activity of p21, and that a combined treatment TNF-alpha/Dox is an effective chemotherapeutic strategy for p53-deficient cancers.     

Characterization of adriamycin-resistant human breast cancer cells which display overexpression of a novel resistance-related membrane protein

Development of multidrug resistance due to overexpression of P-glycoprotein (Pgp), a cell membrane drug efflux pump, occurs commonly during in vitro selections with adriamycin (Adr). Pgp-mediated drug resistance can be overcome by the calcium channel blocker verapamil (Vp), which acts as a competitive inhibitor of drug binding and efflux. In order to identify other mechanisms of Adr resistance, we isolated an Adr-resistant subline by selecting the human breast cancer cell line MCF-7 with incremental increases of Adr in the presence of 10 microgram/ml verapamil. The resultant MCF-7/AdrVp subline is 900-fold resistant to Adr, does not overexpress Pgp, and does not exhibit a decrease in Adr accumulation. It exhibits a unique cross-resistance pattern: high cross-resistance to the potent Adr analogue 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin, lower cross-resistance to the alkylating agent melphalan, and a sensitivity similar to the parental cell line to vinblastine. The levels of glutathione and glutathione S-transferase are similar in the parental line and the Adr-resistant subline. Topoisomerase II-DNA complexes measured by the potassium-sodium dodecyl sulfate precipitation method shows a 2-3 fold decrease in the resistant subline. The MCF-7/AdrVp cells overexpress a novel membrane protein with an apparent molecular mass of 95 kDa. Polyclonal antibodies raised against the P-95 protein demonstrate a correaltion between the level of expression and Adr resistance. Removal of Adr but not verapamil from the selection media results in a decline in P-95 protein levels that parallels a restoration of sensitivity to Adr. Immunohistochemistry demonstrates localization of the P-95 protein on the cell surface. The demonstration of high levels of the protein in clinical samples obtained from patients refractory to Adr suggests that this protein may play a role in clinical drug resistance.     

Altered Expression of Proliferation-Inducing and Proliferation-Inhibiting Genes Might Contribute to Acquired Doxorubicin Resistance in Breast Cancer Cells

This study was designed to investigate the molecular changes that may develop during exposure of breast cancer cells to anticancer agents and that may lead to acquired resistance. We used two breast cancer cell lines, a parental (MCF7/WT) and a doxorubicin-resistant (MCF7/DOX) one. Cell survival, cell cycle distribution and RT-PCR expression level of genes involved in DNA damage response, MDR1, GST and TOPOIIα were measured. MCF7/DOX cells were five-fold more resistant to doxorubicin (DOX) than the MCF7/WT cells. DOX treatment causes arrest of MCF7/DOX cells in G1 and G2 phases of cell cycle whereas MCF7/WT cells were arrested in S-phase. The molecular changes in both cell lines due to DOX treatment could be classified into: (1) the basal level of p53, p21, BRCA1, GST and TOPOIIα mRNA was higher in MCF7/DOX than MCF7/WT. During DOX treatment, the expression level of these genes decreased in both cell lines but the rate of down-regulation was faster in MCF7/WT than MCF7/DOX cells. (2) The expression level of MDR1 was the same in both cell lines but 48 and 72 h of drug treatment, MDR1 disappeared in MCF7/WT but still expressed in MCF7/DOX. (3) There was no change in the expression level of BAX, FAS and BRCA2 in both cell lines. Conclusively, after validation in clinical samples, overexpression of genes like BRCA1, p53, p21, GST, MDR1 and TOPOIIα could be used as a prognostic biomarker for detection of acquired resistance in breast cancer and as therapeutic targets for the improvement of breast cancer treatment strategies.     

Anti-cancer characteristics of mevinolin against three different solid tumor cell lines was not solely p53-dependent

Mevinolin (MVN) has been used clinically for the treatment of hypercholesterolemia with very good tolerance by patients. Based on epidemiological evidences, MVN was suggested strongly for the treatment of neoplasia. Early experimental trials suggested the mixed apoptotic/necrotic cell death pathway was activated in response to MVN exposure. Herein, the cytotoxic profile of MVN was evaluated, compared to the robust and frequently used anti-cancer drug doxorubicin (DOX), against breast (MCF-7), cervical (HeLa) and liver (HepG(2)) transformed cell lines. MVN was showed comparable results in cytotoxic profile with DOX in all tested solid tumor cell lines. In addition, the MVN-induced cytotoxicity was inferred to be multi-factorial and not solely dependent on p53 expression. It was concluded that molecular and genetic assessment of MVN-induced cell death would be useful for developing cancer therapeutic treatments.     

The carotenoid fucoxanthin can sensitize multidrug resistant cancer cells to doxorubicin via induction of apoptosis,inhibition of multidrug resistance proteins and metabolic enzymes

BackgroundMultidrug resistance (MDR) causes failure of doxorubicin therapy of cancer cells, which develops after or during doxorubicin treatment resulting in cross-resistance to structurally and functionally-unrelated other anticancer drugs. MDR is multifactorial phenomenon associated with overexpression of ATP-binding cassette (ABC) transporters, metabolic enzymes, impairment of apoptosis, and alteration of cell cycle checkpoints. The cancer-prevention of the dietary carotenoid; fucoxanthin (FUC) has been extensively explored. Nevertheless, the underlying mechanism of its action is not full elucidated.Hypothesis/PurposeInvestigation of the underlying mechanism of MDR reversal by the dietary carotenoid fucoxanthin (FUC) and its ability to enhance the doxorubicin (DOX) cytotoxicity in resistant breast (MCF-7/ADR), hepatic (HepG-2/ADR), and ovarian (SKOV-3/ADR) cell lines.MethodsThe synergistic interaction of FUC and DOX was evaluated using several techniques, viz.; MTT assay, ABC transporter function assays using FACS and fluorimetry, enzyme activity via spectroscopy and luminescence assays, and apoptosis assay using FACS, and gene expression using RTPCR.ResultsFUC (20 µM) synergistically enhanced the cytotoxicity of DOX and significantly reduced the dose of DOX (FR) in DOX resistant cells (MCF-7/ADR), hepatic (HepG-2/ADR), and ovarian (SKOV-3/ADR) to 8.42-(CI= 0.25), 6.28-(CI= 0.32), and 4.56-fold (CI=0.37) (P<0.001). FUC significantly increased the accumulation of DOX more than verapamil in resistant cells by 2.70, 2.67, and 3.95-fold of untreated cells (p<0.001), respectively. A FUC and DOX combination significantly increased the Rho123 accumulation higher than individual drugs by 2.36-, 2.38-, 1.89-fold verapamil effects in tested cells (p<0.001), respectively. The combination of the FUC and DOX decreased ABCC1, ABCG2, and ABCB1 expression. The FUC and DOX combination increased the levels and activity of caspases (CASP3, CASP8) and p53, while decreased the levels and activity of CYP3A4, GST, and PXR in resistant cancer cells. The combination induced early/late apoptosis to 91.9/5.4% compared with 0.0/0.7% of untreated control.ConclusionOur data suggests a new dietary and therapeutic approach of combining the FUC with DOX to overcome multidrug resistance in cancer cells. However, animal experiments should be conducted to confirm the findings before applying the results into clinical trials.     

Proliferation of human breast cancer cells and anti-cancer action of doxorubicin and vinblastine are independent of PKC-alpha

Protein kinase C (PKC) has been considered for a potential target of anticancer chemotherapy. PKC-alpha has been associated with growth and metastasis of some cancer cells. However, the role of PKC-alpha in human breast cancer cell proliferation and anticancer chemotherapy remains unclear. In this study, we examined whether alterations of PKC-alpha by phorbol esters and PKC inhibitors could affect proliferation of human breast cancer MCF-7 cells and the cytotoxic effect of chemotherapeutic agents. Exposure for 24 h to doxorubicin (DOX) and vinblastine (VIN) caused a concentration-dependent reduction in proliferation of MCF-7 cells. However, these two anticancer drugs altered cellular morphology and growth pattern in distinct manners. Phorbol 12,13-dibutyrate (PDBu, 100 nM), which enhanced activities of PKC-alpha, increased cancer cell proliferation and attenuated VIN (1 microM)-induced cytotoxicity. These effects were not affected in the presence of 10 nM staurosporine. Phorbol myristate acetate (PMA, 100 nM) that completely depleted PKC-alpha also enhanced cancer cell proliferation and attenuated VIN-induced cytotoxicity. Three potent PKC inhibitors, staurosporine (10 nM), chelerythrine (5 microM) and bisindolylmaleimide-I (100 nM), had no significant effect on MCF-7 cell proliferation; staurosporine and chelerythrine, but not bisindolylmaleimide-I, attenuated VIN-induced cytotoxicity. Moreover, neither phorbol esters nor PKC inhibitors had an effect on cytotoxic effects of DOX (1 microM) on MCF-7 cell proliferation. Thus, these data suggest that MCF-7 cell proliferation or the anti-cancer action of DOX and VIN on breast cancer cells is independent of PKC-alpha.     

Chemotherapeutic sensitization of leptomycin B resistant lung cancer cells by pretreatment with doxorubicin

The development of novel targeted therapies has become an important research focus for lung cancer treatment. Our previous study has shown leptomycin B (LMB) significantly inhibited proliferation of lung cancer cells; however, p53 wild type lung cancer cells were resistant to LMB. Therefore, the objective of this study was to develop and evaluate a novel therapeutic strategy to sensitize LMB-resistant lung cancer cells by combining LMB and doxorubicin (DOX). Among the different treatment regimens, pretreatment with DOX (pre-DOX) and subsequent treatment with LMB to A549 cells significantly decreased the 50% inhibitory concentration (IC50) as compared to that of LMB alone (4.4 nM vs. 10.6 nM, P<0.05). Analysis of cell cycle and apoptosis by flow cytometry further confirmed the cytotoxic data. To investigate molecular mechanisms for this drug combination effects, p53 pathways were analyzed by Western blot, and nuclear proteome was evaluated by two dimensional-difference gel electrophoresis (2D-DIGE) and mass spectrometry. In comparison with control groups, the levels of p53, phospho-p53 (ser15), and p21 proteins were significantly increased while phospho-p53 (Thr55) and survivin were significantly decreased after treatments of pre-DOX and LMB (P<0.05). The 2D-DIGE/MS analysis identified that sequestosome 1 (SQSTM1/p62) had a significant increase in pre-DOX and LMB-treated cells (P<0.05). In conclusion, our results suggest that drug-resistant lung cancer cells with p53 wild type could be sensitized to cell death by scheduled combination treatment of DOX and LMB through activating and restoring p53 as well as potentially other signaling pathway(s) involving sequestosome 1.