Human non-small cell carcinoma cell line, A549 was obtained from NCCS, India. Peripheral blood collected from healthy human volunteers with informed consent (Institutional Review Board 1382) was centrifuged over Ficoll-Hypaque density gradient (Amersham Pharmacia, Uppsala, Sweden) to obtain total peripheral blood mono-nuclear cells. Cells were routinely maintained in DMEM supplemented with 10% heat inactivated fetal bovine serum (Lonza, NH, USA), L-glutamine (2 mM), sodium pyruvate (100 μg/ml), non-essential amino acids (100 μM), streptomycin (100 μg/ml), penicillin (50 U/ml; Invitogen, CA, USA) at 37°C in a humidified 5% CO₂ incubator. Cells were maintained in an exponential growth phase for all experiments. Viable cell numbers were determined by trypan-blue exclusion test.

Placebo and sulphur 6C, 30C or 200C were procured from Hahnemann Publishing Co., India. Cells were treated with sulphur/placebo of potencies 6, 30 or 200C exposure at the different concentration (10, 15, 20 and 30 μl/ml) for different time-points (6, 12, 24, 36 and 48 h) to select the optimum time required for cell killing. To understand the sequence of events leading to apoptosis, cancer cells were treated with mitochondrial pore inhibitor CsA (25 μM; Merck, Germany) for 1 h prior to incubation with sulphur.

For the determination of cell death, cells were stained with 7AAD and Annexin V-FITC and analyzed on flow cytometry (FACS Verse, BD Biosciences). Electronic compensation of the instrument was done to exclude overlapping of the emission spectra. A total of 10,000 events were acquired for analysis using CellQuest software. For the assessment of mitochondrial transmembrane potential, cells were stained with potentially-sensitive dye Dihexyloxacarbonicao cyanine (DiOC₆, Merck, Germany) during the last 30 min of treatment at 37°C in the dark. Fluorescence of retained DiOC₆ was determined flow cytometrically using logarithmic amplification by CellQuest software (BD Biosciences) (31).

Chromatin condensation and nuclear fragmentation was analyzed using the standard protocol. Briefly, cells were grown on coverslips, fixed with 3% p-formaldehyde for 10 min and then permeabilized with 0.1% Triton X-100 for 5 min. Cells were then incubated with 4′6-diamidino-2-phenyl-indole (DAPI; BD Pharmingen, CA, USA). The morphology of the cell nuclei was visualized using a fluorescence microscope (Leitz microscope fitted with epifluorescence illuminator through a 60× aperture oil immersion lens, Carl Zeiss, Germany). For fluorescence imaging, cells growing on a cover slip were fixed with 3% p-formaldehyde and were stained with anti-p53 and anti-p65NFκB antibodies (SantaCruz, CA, USA), after permeabilization with Triton X-100, followed by FITC and TRITC conjugated secondary antibodies, respectively and visualized with confocal microscope (Carl Zeiss, Germany) (13,37).

The expression constructs pcDNA3.0/HA-tagged IκBα-32A/36A [IκBα super-repressor (IκBα-SR), a kind gift from Dr J. Didonato, The Cleveland Clinic Foundation], pcDNA3.1-p65NFκB/p53/Bcl-2 overexpression plasmids (2 μg/million cells) were introduced into exponentially growing cancer cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the protocol provided by the manufacturer. In a similar manner, p53 expression was knocked down in A549 cells using p53-shRNA (Santa Cruz) and Lipofectamine 2000 (Invitrogen). Stably expressing clones were isolated by limiting dilution and selected with G418 sulphate (Cellgro) at a concentration of 400 μg/ml and cells surviving this treatment were cloned and screened by western blot analysis with specific antibody. A549 cells were transfected with 300 pmol of Bcl-2-/Bax-/control-ds-siRNA (Santa Cruz) and Lipofectamine 2000 separately for 12 h. The levels of respective proteins were estimated by western blotting (37).

To obtain whole cell lysates, cells were homogenized in lysis buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM Na-EDTA, 1 mM Na-EGTA and 1 mM DTT) supplemented with protease and phosphatase inhibitor cocktails. Mitochondrial and cytosolic fractions were prepared according to Lahiry et al (31). For direct western blot analysis, a total of 50 μg of protein was resolved using SDS-PAGE and transferred to nitrocellulose membrane for western blotting using required antibodies e.g., anti-caspase-9, anti-caspase-3, anti-p53 (DO-1), anti-p65NFκB, anti-IκBα, anti-Bcl-2, anti-Bax (N-20), anti-cytochrome c and anti-p300. Thereafter proteins of interest were visualized by chemiluminiscence. Equivalent protein loading in cytosolic, nuclear and mitochondrial fractions were verified using anti-α-actin/ histone H1/MnSOD antibodies (Santa Cruz, CA, USA) respectively.

For the determination of direct interaction between two proteins, co-immunoprecipitation technique was employed (38,39). p53–p300 and p65NFκB-p300 interaction was determined by co-immunoprecipitation. Samples (300 μg of protein from the total lysate) were incubated at 4°C overnight with anti-p300/-IgG antibody and then incubated for 2 h at 4°C with protein A-Sepharose. Immunocomplexes were washed of unbound proteins with cold TBS with protease inhibitors, and pelleted beads were boiled for 5 min in SDS-PAGE sample buffer. The immunoprecipitated proteins were resolved on SDS-PAGE and analyzed by western blotting for detection of associated proteins. Equal protein loading was confirmed using anti-histone H1 antibody.

Total RNA (2 μg) each from untreated, placebo-/sulphur-treated NSCLC cells was extracted by TRIzol (Invitrogen) and was reverse transcribed and then subjected to PCR with enzymes and reagents of the RTplusPCR System (Eppendorf, Hamburg, Germany) using GeneAmpPCR 2720 (Applied Biosystems, Foster City, CA, USA) (40). The cDNAs were amplified with primers specific for Bax (5′-GGAATTCCAAGAAGCTGAGCGAGTGT-3′/ 5′-GGAATTCTTCTTCCAGATGGTGAGCGAG-3′), Bcl-2 (5′-CCTGTGCCACCATGTGTCCATC-3′/5′-GCTGAGAACA GGGTCTTCAGAGAC-3′) and GAPDH (internal control: 5′-TGATGACATCAAGAAGGTGGTGAAG-3′/5′-TCCTTGG AGGCCATGTAGGCCAT-3′).

ChIP assays were carried out for identification of p53 and p65NFκB binding region on Bax and Bcl-2-promoters respectively, using a ChIP assay kit (Millipore) according to the manufacturer’s instructions. PCR assay was performed using primer sets as follows: Bcl-2 forward primer 5′-GATTCCTGCGGATTGACA TTTC-3′, Bcl-2 reverse primer 5′-CATCAATCTTCAGCACTC TCC-3′; Bax forward primer 5′-TCAGCACAGATTAGTTT CTG-3′, Bax reverse primer 5′-GGGATTACAGGCATGAG CTA-3′. Extracted DNA (2 μl) was used for 45 cycles of amplification in 5 μl of reaction mixture under the following conditions: 95°C for 30 sec, 56°C for 30 sec and 72°C for 60 sec. The PCR products were analysed by 2% agarose gel electrophoresis (41,42).

Values are shown as standard error of mean, except when otherwise indicated. Data were analyzed and, significance (P<0.05) of the differences between mean values was determined by a Student’s t-test.

The effect of sulphur, a homeopathic drug, on the viability of human NSCLC cell line (A549) and normal peripheral blood mononuclear cell (PBMC) was examined at different potencies of sulphur, i.e., 6C, 30C, 200C, where for each potency a differential dose of 0–30 μl/ml was applied (Fig. 1A and B). The percent cell death was scored by trypan-blue dye-exclusion assay. It was observed that among all the potencies of sulphur, 30C potency resulted in most significant decrease (p<0.001) in cell viability (Fig. 1A). Moreover, at 20 μl/ml dose of 30C, the percent A549 cell death reached its optimum (Fig. 1A) while under the same conditions the PBMC viability was found to be >90% (Fig. 1B). These results indicating the better efficacy of 20 μl/ml dose of 30C sulphur in A549 cell killing with minimum toxicity led us to perform all further experiments using this particular potency and dose of sulphur. The time-dependent effect of sulphur 30C (20 μl/ml), in comparison to placebo, on A549 and PBMC cells was examined at different time intervals (0–48 h) and percentage cell death was assessed. Sulphur 30C, at a concentration of 20 μl/ml, exerted time-dependent significant death in A549 cells (Fig. 1C). However, significant cell death in PBMCs was noted from 24 h onwards following the treatment (Fig. 1D).

Next, to confirm the nature of cell death as apoptosis, we utilized double labelling techniques using Annexin V-FITC/7-AAD to distinguish between apoptotic and necrotic cells. Our flow cytometric data demonstrated that in comparison to placebo-treated A549 cells, sulphur-treated unfixed A549 cells showed Annexin V-FITC binding with minimum 7-AAD binding (Fig. 1E) indicating that the mode of cell death was apoptosis but not necrosis. These findings were re-confirmed by the development of nuclear blebbing as evidenced by DAPI-stained fluorescent images of sulphur-treated A549 cells (Fig. 1F). These data together supported the notion that sulphur 30C asserts apoptogenic effect in the NSCLC A549 cells.

Since p65NFκB has been reported to be globally involved in survival of cancer cells (27,28,43), we examined whether sulphur suppresses this pathway to combat NFκB-mediated survival to induce apoptosis in drug-resistant NSCLC cells. Our search revealed that sulphur treatment efficiently blocked nuclear translocation of NFκB in NSCLC cells as observed by both western blotting (Fig. 2A) and confocal imaging experiments (Fig. 2B). In addition, the mRNA and protein levels of NFκB-target gene, Bcl-2, was found to be downregulated by sulphur treatment in NSCLC cells (Fig. 2C). We further observed that transfecting NSCLC cells with super repressor IκBα-SR-cDNA decreased Bcl-2 followed by significant apoptosis in response to sulphur (Figs. 2D and E). On the other hand NSCLC cells expressing p65NFκB-cDNA manifested enhanced Bcl-2 with significant resistance upon sulphur exposure (Fig. 2D and E). The anti-apoptotic role of NFκB-dependent Bcl-2 upregulation was re-confirmed by evaluating response of Bcl-2-engineered cells towards sulphur treatment. Overexpression of Bcl-2 in NSCLC cells bestowed them with survival advantage upon sulphur treatment, whereas transfection with Bcl-2-siRNA efficiently enhanced sulphur-induced apoptosis (Fig. 2F). Collectively, these results confirmed that p65NFκB activation and subsequent Bcl-2 upregulation were primarily involved in survival of NSCLC cells which upon inhibition by sulphur induced apoptosis in these cells.

Since inhibition of p65NFκB activity in NSCLC cells induced a powerful apoptotic response, we predicted the involvement of the cellular apoptotic proteins during sulphur-induced apoptosis. We evaluated the status of apoptotic proteases, i.e., caspase-9 and caspase-3, respectively, in response to sulphur treatment. It was noted that sulphur treatment significantly upregulated levels of cleaved caspase-9 and caspase-3 in A549 cells (Fig. 3A). Since tumor suppressor protein p53 plays an important role in canonical apoptotic pathway, the above results tempted us to compare the p53 activation status upon sulphur exposure in NSCLC cells. Results of Fig. 3B left panel revealed that sulphur induced p53 expression in A549 cells when compared to untreated cells. These results were further confirmed by confocal microscopy, the results of which not only demonstrated accumulation of p53 protein but also its translocation from cytosol to nucleus of sulphur-exposed A549 cells (Fig. 3B, middle panel). Furthermore, findings of western blot analysis verified greater translocation of p53 from cytosol to nucleus upon sulphur exposure in A549 cells (Fig. 3B right panel). It is acknowledged that p53 when localized in nucleus transactivates its downstream target genes (31). We, therefore, next assessed the status of its trans-activated gene product, i.e., Bax, by western blot and RT-PCR analyses in sulphur-treated and untreated A549 cells. Our results re-established that in comparison to un-/placebo-treated tumor cells, sulphur-exposed cells showed increase in the levels of p53 trans-activated gene product Bax (Fig. 3C) Downstream of Bax, increase in cytosolic cytochrome c with its concomitant decrease in mitochondria (Fig. 3D) were observed and also significant mitochondrial transmembrane potential (MTP) loss is observed in sulphur-exposed A549 cells as compared to control and placebo-treated A549 cells (Fig. 3E).

Next, A549 cells were transfected with Bax-siRNA or pre-treated with cyclosporine A (CsA), mitochondrial pore formation blocker, prior to sulphur treatment for validation of the involvement of Bax in mitochondria cascade-mediated NSCLC apoptosis. Silencing Bax, or CsA pre-treatment significantly decreased percent apoptosis of NSCLC cells and also down-modulated the expression levels of caspase-9 and caspase-3 (Fig. 3F). Taken together these findings validated the contribution of Bax in sulphur-induced A549 cell apoptosis via mitochondrial death cascade.

Next, we verified whether sulphur-induced cancer cell death is p53-dependent or not. To this end, human cancer cell lines with differential p53 status, e.g., wild-type p53-expressing and p53-shRNA-transfected A549 cells were tested for sulphur-dependent apoptosis by scoring the number of Annexin V-positive cells flow cytometrically (Fig. 4A). Interestingly, sulphur 30C at 20 μl/ml dose significantly (p<0.001) induced apoptosis in wild-type p53-expressing A549 cells. The apoptogenic insult asserted by sulphur after minimization of placebo effect in A549 cells were 31%, while p53-knockdown cells resisted such insult (Fig. 4A). These results indicated the contribution of functional p53 in sulphur-induced cancer cell apoptosis.

In parallel experiment when A549 cells were transfected with p53-cDNA, p53 expression though increased, Bax expression level failed to reach that of sulphur-treated A549 cells (Fig. 4B). These findings revealed that increasing p53 levels alone in NSCLC cells failed to restore p53 transcriptional functions and to induce apoptosis (Fig. 4C). This raised the possibility of the involvement of p53 transcriptional ‘inhibitor(s)’ in NSCLC cells that somehow opposed p53-dependent transcription of apoptotic genes. Sulphur, on the other hand, by restraining this inhibitor might have activated the p53-transcriptional program. Since our previous results (Fig. 2A and B) have demonstrated sulphur-induced inhibition of NFκB activation, we hypothesized that activated NFκB might be blocking p53-dependent apoptotic program in NSCLC cells. To confirm this hypothesis we utilized IκBα-SR-cDNA transfected cells and checked p53-dependent execution of apoptosis in these cells upon transfection with p53-cDNA. Indeed these transfectants displayed robust p53 induction along with upregulation of Bax (Fig. 4B). Activation of caspase-3 in these cells (Fig. 4B) finally confirmed that NFκB intervened the functioning of p53-dependent apoptotic program.

All these results together signified that sulphur by inhibiting p65NFκB-governed survival signalling skewed the cellular microenvironment in favor of p53-transcriptional activation to result in apoptosis.

We next attempted to unveil the detail mechanisms underlying NFκB-mediated inhibition of p53 transcription functions. Recent studies indicate that the transcriptional activity of p53 is regulated by its interaction with the transcriptional co-activator p300 (27,31). To verify the effects of sulphur on p53–p300 cross-talk, if any, we immunoprecipitated nuclear p300 in NSCLC cells treated with placebo, or sulphur and verified its interaction with p53 by western blotting. It was observed that in contrast to placebo, sulphur induced p53–p300 interaction (Fig. 5A). Consistently, it was further observed by ChIP analysis that sulphur treatment in NSCLC cells enhanced p53 binding on Bax promoter, which is a pre-requisite for transcriptional activation of Bax (Fig. 5B). This subsequently enabled p53-dependent apoptosis as observed earlier (Fig. 3A). Since, sulphur triggered p53–p300 interaction in cells where p65NFκB activation was not taking place, we proposed that nuclear translocation of p65NFκB in untreated p53-cDNA transfected A549 cells might have sequestered p300 thereby abridging p53–p300 cross-talk. As anticipated, these untreated p53-cDNA transfected A549 cells manifested significant p65NFκB-bound p300 in their nuclear lysates (Fig. 5C). Furthermore, upon genetically perturbing nuclear translocation of p65NFκB in these p53-cDNA trasfected A549 cells, significant increase in p53–p300 interaction was observed with concomitant decrease in p65NFκB-bound p300 (Fig. 5D). Interestingly sulphur treatment inhibited p65NFκB-p300 cross-talk (Fig. 5E) thereby preventing p65NFκB binding on Bcl-2 promoter to initiate p65NFκB-mediated Bcl-2 transcription (Fig. 5F). These results indicate a competition between NFκB and p53 for availing p300, and depending on the relative availability, the winner, and the fate of the cells are decided.

In summary, the above results conclude that in drug-resistant NSCLC cells, p65NFκB competes with p53 for the transcriptional co-activator p300 thereby inhibiting the apoptotic program and upregulates the survival-machinery of the cell. On the other hand, by inhibiting p65NFκB, sulphur censored the survival pathway thereby making p300 available for p53 interaction to ensure the transcription of pro-apoptotic protein Bax for effective induction of apoptosis in otherwise resistant NSCLC cells (Fig. 6).