Polyphenols Sensitization Potentiates Susceptibility of MCF-7 and MDA MB-231 Cells to Centchroman

Polyphenols as “sensitizers” together with cytotoxic drugs as “inducers” cooperate to trigger apoptosis in various cancer cells. Hence, their combination having similar mode of mechanism may be a novel approach to enhance the efficacy of inducers. Additionally, this will also enable to achieve the physiological concentrations facilitating significant increase in the activity at concentrations which the compound can individually provide. Here we propose that polyphenols (Resveratrol (RES) and Curcumin (CUR)) pre-treatment may sensitize MCF-7/MDA MB-231 (Human Breast Cancer Cells, HBCCs) to Centchroman (CC, antineoplastic agent). 6 h pre-treated cells with 10 µM RES/CUR and 100 µM RES/30 µM CUR doses, followed by 10 µM CC for 18 h were investigated for Ser-167 ER-phosphorylation, cell cycle arrest, redox homeostasis, stress activated protein kinase (SAPKs: JNK and p38 MAPK) pathways and downstream apoptosis effectors. Low dose RES/CUR enhances the CC action through ROS mediated JNK/p38 as well as mitochondrial pathway in MCF-7 cells. However, RES/CUR sensitization enhanced apoptosis in p53 mutant MDA MB-231 cells without/with involvement of ROS mediated JNK/p38 adjunct to Caspase-9. Contrarily, through high dose sensitization in CC treated cells, the parameters remained unaltered as in polyphenols alone. We conclude that differential sensitization of HBCCs with low dose polyphenol augments apoptotic efficacy of CC. This may offer a novel approach to achieve enhanced action of CC with concomitant reduction of side effects enabling improved management of hormone-dependent breast cancer

S-phase fraction as a useful marker for prognosis and therapeutic response in patients with aplastic anemia

BACKGROUND: The functional definition of aplastic anemia (AA) is the failure of hematopoietic stem cells to proliferate. The aim of the present study was to analyze the S-phase fraction (SPF) (proliferative activity) in patients with AA at diagnosis to explore its relationship with disease characteristics and its value in discriminating among patients with different prognoses. We also investigated whether the SPF value influenced the response to immunosuppressive therapy in AA patients. PATIENTS AND METHODS: The analysis of SPF at the time of diagnosis was carried out by flow cytometry on peripheral blood samples from 53 consecutive patients with AA and 30 age- and sex-matched controls. All patients were given cyclosporine and followed up periodically to determine response to therapy. RESULTS: Based on the median SPF, AA patients were divided into two groups: patients with SPF ≤0.59% (n=27) and patients with SPF >0.59% (n=26). An SPF >0.59% was associated with advanced age (P=.02) and elevated serum LDH level (P=.01). Patients with an SPF >0.59% also had a higher incidence of paroxysmal nocturnal hemoglobinuria and cytogenetic abnormalities. During a median follow-up of 18 months, 3.7% of patients with SPF ≤0.59 and 11.5% of patients with SPF >0.59% developed dysplasia and one patient with SPF >0.59% converted into AML. A significantly higher (P=.018) overall response rate of 53.9% was found in patients with SPF >0.59% versus 22.2% of patients with SPF ≤0.59% at 6 months. CONCLUSIONS: Independently of the peripheral blood count, the SPF at diagnosis may provide information on the expected response to immunosuppressive therapy and the propensity for disease to evolve into MDS/AML. Hence, SPF may serve as an early indicator for the evolution of MDS/AML in patients with AA and thus contribute to therapeutic decisions

Cell-Cycle analysis of RES, CUR and CC treated MCF-7 cells (Time-dependent and sensitized).

<p>Briefly, 0.2×10⁶ cells were pre-cultured for 48 h in phenol red-free DMEM then exposed for 3, 6 and 24 h with 10 µM/100 µM RES {A (MCF-7), B (MDA MB-231)}, 10 µM CC (C), 10 µM/30 µM CUR {D (MCF-7), E (MDA MB-231)} or preconditioned cells with polyphenols (RES: 10/100 µM and CUR: 10/30 µM) for 6 h and continued with CC (10 µM) for next 18 h (total of 24 h). Following incubations the MCF-7 (F) and MDA MB-231 (G) cells were harvested, permeabilized, stained with Propidium Iodide (40 µg/mL) and analyzed by Flow Cytometer (employing Modfit Software); % CV <10. Data shown are the mean ± S.E. of one of the three similar experiments each performed in triplicate. * P<0.05; ** P<0.01; *** P<0.001, calculated compared to control. $P<0.05;$$P<0.01;$$$ P<0.001 with respect to individual CC dose and # P<0.05; ## P<0.01; ### P<0.001 with respect to individual RES dose.</p

Ser-167 phosphorylation of AF-1 region of ER α.

<p>3×10⁶ MCF-7/MDA MB-231 cells were exposed to CC (10 µM)/polyphenols <i>per se</i> (RES: 10/100 µM and CUR: 10/30 µM) or sensitized with low/high dose polyphenols for 6 h and continued with CC (10 µM) for next 18 h. 50 µg of the whole cell lysate was separated on a 10% SDS-PAGE, probed with respective antisera following transfer to a nitrocellulose membrane with immunoblotting using β-actin as control. Data shown is one of three similar experiments each performed in triplicate.</p

Time-dependent ROS-generation and Mitochondrial Membrane Potential (ΔΨm).

<p>0.2×10⁶ MCF-7/MDA MB-231 cells were plated in 6-well plates, treated time-dependently (3, 6 and 24 h) with CC (10 µM) and polyphenols <i>per se</i> {(RES: 10/100 µM and CUR: 10/30 µM; A (ROS-generation) and B (ΔΨm)}; preconditioned cells with low and high dose polyphenols for 6 h and continued with CC (10 µM) for next 18 h {C (ROS-generation) and D (ΔΨm)}. Cells were then rinsed with PBS and then placed in a preheated 37°C fluorimeter for time-dependent measurement of generation of H₂O₂ (DCFDA). ΔΨm was determined on a Beckton–Dickinson Fluorescence Activated Cell Sorter. Flow Cytometry analysis of MCF-7 and MDA MB-231 cells stained with DCFDA was performed to confirm ROS results using N-Acetyl-l-Cysteine (L-NAC, 5 mM) 1 hr prior to treatment (E). Data shown are the Mean ± S.E. of one of the three similar experiments each performed in triplicate. * P<0.05; ** P<0.01; *** P<0.001, calculated compared to control. $P<0.05;$$P<0.01;$$$ P<0.001 with respect to individual CC dose and # P<0.05; ## P<0.01; ### P<0.001 with respect to individual RES dose.</p

Time-dependent JNK, p38 and p53 pathway inhibition studies induced by CC and polyphenols alone and JNK/p38-dependent apoptosis in sensitized cells.

<p>10⁴ MCF-7 (A)/MDA MB-231 (B) cells were pre-cultured for 48 h in phenol red-free DMEM then exposed to CC (10 µM)/polyphenols <i>per se</i> (RES: 10/100 µM and CUR: 10/30 µM) for MTT analysis. Percentage inhibition was determined per the formula {(Absorbance of Control – Absorbance of drug treated cells without and with inhibitor)/Absorbance of Control cells} ×100. For inhibitor experiments through Flow Cytometry, 0.2×10⁶ MCF-7 (C)/MDA MB-231 (D) cells were pre-cultured for 48 h in phenol red-free DMEM, exposed to CC (10 µM)/polyphenols <i>per se</i> (RES: 10/100 µM and CUR: 10/30 µM) or preconditioned with low/high dose polyphenols for 6 h and continued with CC (10 µM) for next 18 h. JNK (10 µM SP600125), p38 pathway (1 µM SB 203580), Pifithrin (15 µM Pif) and zVAD-fmk (30 µM) inhibitors were added during the last 2 hours prior to CC exposure, followed by the analysis of the Sub-G0/G1 fraction by FACS. Each data point is the Mean ± S.E. of one of three similar experiments each performed in triplicate. * P<0.05, ** P<0.01, *** P<0.001 drugs compared to drug treatment without inhibitor.</p

JNK/p38-dependent modulation of expression of Antioxidant enzymes, p53 and apoptogenic factors in MCF-7 cells`1.

<p>For expression analysis, 3×10⁶ MCF-7 cells were pre-cultured for 48 h in phenol red-free DMEM and exposed to CC (10 µM) and polyphenols <i>per se</i> {RES: 10/100 µM (A & B) and CUR: 10/30 µM (C & D)} or sensitized with low/high dose polyphenols for 6 h and continued with CC (10 µM) for next 18 h. Using 10 µM of SP600125 (JNK inhibitor)/1 µM of SB 203580 (p38 inhibitor) 2 hours prior to CC exposure, 50 µg of the whole cell lysate was separated on a 10% SDS-PAGE gel, probed with respective antisera following transfer to a nitrocellulose membrane and subsequent immunoblotting. β-actin was used as a protein loading control. (A) RES/CC+RES without and with SP600125; (B) RES/CC+RES without and with SB203580; (C) CUR/CC+CUR without and with SP600125; (D) CUR/CC+CUR without and with SB203580. However, for c-Jun and phospho ATF-2 detection, immunoprecipitation and detection was carried out as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037736#s2" target="_blank">Materials and Methods</a>. Data shown is representative of one of the three similar experiments each performed in triplicate.</p