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eader at the wavelength of 570 nm. The percent of cell viability related to each drug concentration was estimated in relation to the untreated sample. All assays were done at least three times unless stated otherwise. Apoptosis quantification After washing <strong>10</strong> 6 cells with PBS, cell pellets were re-suspended in <strong>10</strong>0 µl of ready to use Annexin/PI buffer (20 µl of each Annexin and PI buffer in 1 ml incubation buffer) for <strong>10</strong>-15 min at 25˚C. Samples were then diluted in 500 µl of incubation buffer and analyzed by flow cytometry (Partech Pass, USA) using FloMax software. Cell cycle analysis 5×<strong>10</strong> 5 drug treated cells were incubated with DAPI solution (<strong>10</strong> µg/ml and 6% Triton X-<strong>10</strong>0 in PBS) for 30 min in the dark at 4ºC. Using a flow cytometer fluorescent emission of applied indicator was detected (excitation and emission wavelength of 359 nm and 461 nm, respectively) and the analysis was performed using FloMax software. Morphological studies of the apoptotic cells Cells were cultured on cover slips coated with Poly L-lysine and exposed to drugs for 48 h. Following staining with Annexin V-FITC (20 µg/ml) and PI (20 µg/ml) in the dark for <strong>10</strong>-15 min, samples were examined using a fluorescent microscope (Carl Zeiss-Germany) using 450-500 nm excitation and 515-565 nm emission filters. Measurement of caspase-3 activity Following drug treatments, cells were harvested and incubated in lysis buffer on ice for 1 minute. After centrifugation, sample supernatants were used for caspase-3 activity measurements using AC- DEVED-AFC fluorescent substrate as recommended by the supplier. The concentration of enzyme-released AFC was estimated using fluorospectrophotometer (HITACHI model MPF4- Japan) at 400 nm excitation and 505 nm emission wavelengths. DNA laddering assay 2×<strong>10</strong> 6 drug treated cells were lysed with an equal volume of binding/lysis buffer for <strong>10</strong> minutes at 15- 25 º C. The obtained extract was processed as recommended by the supplier. Electrophoresis of the samples in 1% agarose gel at 75 volt for 90 minutes revealed DNA cleavage pattern of cells Sulfabenzamide promotes autophagic cell death 43 relative to positive control (DNA extracted prepared from U937 cells incubated 3h with 4 µM camptothecin). Preparation of total RNA, cDNA synthesis and real time RT-PCR Total RNA was purified using the RNeasy Qiagen kit according to the manufacturer’s recommendation. First strand cDNA was generated using RevertAidTM M-MuLV reverse transcriptase and 5µg of RNA with random hexamer primers. Real time quantitative RT-PCR was performed using the QuantiFast SYBR Green PCR Master Mix under the following program: 95˚C for 5 min followed by 40 cycles (95˚C for <strong>10</strong> sec, annealing for 25 sec and extension at 72˚C for 30 sec). Analysis was done using Corbett rotor-gene 6000 software based on the comparative Ct method (or ΔΔCt method). The relative amount of target materials was quantified compared to the reference gene (GAPDH). Primers were prepared by TAG (Copenhagen, Denmark) and were used to amplify specific regions of cDNA as listed in Table1. Statistical analysis For all methods statistical analysis were performed by the SPSS version 16 and Excel 2007 softwares. Statistical analysis for MTT assay, flow cytometry, caspase-3 activity were performed by one way ANOVA and real time RT-PCR methods were carried out by t-test. All results are presented as mean ± standard deviation (p< 0.05 was considered statistically significant). Results Sulfabenzamide inhibits the proliferation of T-47D cells The MTT assay was used to evaluate the viability of T-47D cells incubated with different concentrations of sulfabenzamide (0.0-20 mM) or doxorubicin (0.0-0.6 µM) after 24, 48 and 72 h (chemical structures are shown in Figure 1A). We checked toxic effects of doxorubicin on T-47D since it had been reported that its anticancer effects on different cell types exerts through distinct cellular processes (apoptosis or cell cycle arrest). Thus, it could be utilized as a control in our experiments. The 50% growth inhibition (LC50) concentration for sulfabenzamide and doxorubicin after 48 h, were calculated as <strong>10</strong>.8 mM and 0.33 µM, respectively, and utilized in the following experiments (Figure 1B).
44 Raziye MOHAMMADPOUR et al. Table 1. List of primers. Forward and reverse primer pairs for PTEN gene were designed to amplify a region which could not anneal to PTEN pseudogene. Primer for p53 was designed for the mutant form present in T-47D cells. Gene Accession number primers F: CCAGGTGGTCTCCTCTGACTTCAACAG PCR product(bp) GAPDH NC_000012.11 R: AGGGTCTCTCTCTTCTTCCTCTTGTGCTCT F: GTGAGATATGGTTTGAATATGAAGGC 218 ATG5 NC_000006.11 R: CTCTTAAAATGTACTGTGATGTTCCAA F: GGAGAGGAGCCATTTATTGAAACT 122 beclin1 NC_000017.<strong>10</strong> R: AGAGTGAAGCTGTTGGCACTTTCTG F: CTTGGATTGGTGGGATGTTTC <strong>10</strong>4 DRAM NC_000012.11 R: GATGATGGACTGTAGGAGCGTGT F: CCAGATGGAAAGACGTTTTTGTG 135 AKT1 NC_000014.8 R: GAGAACAAACTGGATGAAATAAA F: CTGCGGAAGGAAGTCATCATTGC <strong>10</strong>6 AKT2 NC_000019.9 R: CGGTCGTGGGTCTGGAAGGCATAC F: CAAACTTTTTCAGAGGGGATCG 125 caspase-3 NC_000004.11 R: GCATACTGTTTCAGCATGGCAC F: AAGAAGCTGAGCGAGTGTC 261 bax NC_000019.9 R: GGCCCCAGTTGAAGTTGC F: ATGGAACTAACTATGTTGGACTATG 157 cyclinB1 NC_000005.9 R: AGTATATGACAGGTAATGTTGTAGAGT F: AGGGGGAAACACCAGAATCAAGTG 138 bcl-2 NC_000018.9 R: CCCAGAGAAAGAAGAGGAGTTATAA F: GGTCTTGTGGACAGTAGTTTGCC 113 AIF NC_000023.<strong>10</strong> R: TCTCACTCTCTGATCGGATACCA F:CCTGTGCAGCTGTGGGTTGATTT 115 p53 NC_000017.<strong>10</strong> R: AGGAGGGGCCAGACCATCGCTAT F: TTGGAGTCCCGATTTCAGAG 150 DFF40 NC_000001.<strong>10</strong> R: CTGTCGAAGTAGCTGCCATTG F:TTCTGTGTCTACCTTCCAATACTA 194 DFF45 NC_000001.<strong>10</strong> R:CTGTCTGTTTCATCTACATCAAAG F: TAACATTAGTCTGGATGGTGTAGA 127 PARP1 NC_000001.<strong>10</strong> R: TTACCTGAGCAATATCATAGACAAT F: TGGCATTACAGACATCTTTAGTTT 113 DNA-PK NC_000008.<strong>10</strong> R: ACTTTAGGATTTCTTCTCTACATTCA F: TGGCTAAGTGAAGATGACAATCATG 111 PTEN NC_0000<strong>10</strong>.<strong>10</strong> R: GCACATATCATTACACCAGTTCGT 81
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Page 10 and 11: dimethoxyphenol (2, 6-DMP). Kinetic
Page 12 and 13: pharmaceutical importance that has
Page 14 and 15: Gianfreda L, Xu F, Bollag JM. Lacca
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Page 67 and 68: 62 Muhammad IRFAN et al. Figure 9.
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Page 71 and 72: 66 Abstract: A brief, informative a
Page 73 and 74: 68 may ask one referee to comment o

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