in view of growing awareness of the side - effects of some orthodox medicines , people are turning more towards alternative medicines with fewer and less - toxic side - effects , and homeopathy has become a major complementary and alternative medicine ( cam ) in many countries today . in homeopathy ,
microdoses of very high dilutions ( potentized ) of natural substances are generally preferred over mother tinctures ( crude extracts ) [ 2 - 4 ] for stronger and longer - effects .
the initial drug substance is generally dissolved in an aqueous solution of ethanol ( mostly 70% ) and is potentized in gradual steps of dilution with agitation or succussion . on a centesimal scale , when 1 ml of mother tincture is diluted with 99 ml of an aqueous solution of ethanol ( vehicle of drug ) and given 10 mechanical jerks , potency 1c is produced .
when 1 ml of 1c is again diluted with 99 ml of an aqueous solution of ethanol and given 10 jerks , the potency 2c is produced , and so on .
therefore , when the drug has attained potency 12c , it has been diluted to 10 ( beyond avogadro s limit ) , and the existence of even a single molecule of the original drug substance becomes highly improbable .
although some researchers have demonstrated the existence of nanoparticles of the original drug in such ultra - highly diluted homeopathic drugs [ 5 , 6 ] , the efficacy is often questioned by rationalists , as the precise mechanism of drug action has still not been firmly established .
therefore , we became interested in the study of any perceivable differences in the actions between dilutions below avogadro s limit ( 6c , diluted 10 times ) and above avogadro s limit ( 30c , diluted 10 times ) in living cells , in vitro , and we attempted to understand the possible signalling pathway of their action .
non - small cell lung cancer ( nsclc ) is the most prevalent accounting for - 80% of all lung cancer cases .
in addition , benzo[a]pyrene is one of the major polycyclic aromatic hydrocarbons found in cigarette smoke and is responsible for inducing lung tumours in smokers .
patients with lung cancer are commonly treated with conventional modalities including chemotherapy , radiation therapy , etc . which also affect normal cells .
, are now gaining importance in the cure / amelioration of many difficult- to - cure diseases including cancer .
first , do the two potentized homeopathic drugs , condurango 6c and 30c show any ability to induce apoptosis in nsclc , and do they act via reactive oxygen species ( ros ) generation and mitochondrial membrane potential ( mmp)-depolarization .
second , dose if condurango 30c have more apoptosis - inducing ability than condurango 6c .
nci - h460 ( h460 ) human nsclc cells were procured from national centre for cell science ( nccs ) , pune , india and cultured in rpmi-1640 media , supplemented with 10%-fetal bovine serum ( fbs ) and 1%-antibiotic - antimicotic solution .
h460 cells ( 110 /well ) were treated with different concentrations of condurango 6c and 30c ( 0.5l/100l media- 5l/100l media ) and with placebos ( successed 70% alcohol - vehicle for the drugs ) as a control for 24 hours and 48 hours .
the concentrations at which both the drugs showed nearly 50% cell death were determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) assays .
h460 cells were also treated with half maximal inhibitory concentration ( ic50 ) doses of condurango 6c and 30c and were compared against placebo - treated and untreated cells . after 24 hours and 48 hours of treatment , the cells were observed and photographed under an inverted phase - contrast microscope ( axiscope plus 2 , zeiss , germany ) . for further confirmation of morphological changes , if any , a scanning electron microscopy ( sem ) study was done   using an s530-hitachi scanning electron microscope .
reactive oxygen species ( ros)-accumulation is generally known to occur at early hours of apoptosis .
cells were treated with condurango 6c and 30c ( ic50 doses ) and with their respective placebos for 2 hours , 6 hours , 12 hours , 18 hours , and 24 hours , to estimate the specific time - point at which maximum ros accumulated . after treatment
, cells were incubated with 2,7-dichlorodihydrofluorescein diacetate ( h2 dcfda ) ( 5 mm ) and ros was estimated by using fluorimetry ( perkinelmer , usa ) .
fluorescence microscopy was done at the specific time - point(s ) when ros - generation was maximum .
the changes in mmp were recorded at 2 hours , 6 hours , 12 hours , 18 hours , and 24 hours of treatment with ic50 doses of both drugs were compared against drug - untreated cells by using rhodamine-123 and the cells were analyzed by using fluorescence microscope ( leica , dmls ) .
changes in mmp were also measured using flowcytometry ( facs , aria iii , bd bioscience ) at the specific time - point(s ) at which the mmp showed the maximum decrease .
cells were stained separately with 4,6-diamidino-2-phenylindole ( dapi ) ( 10gml ) and acridine orange / ethidium bromide ( ao / eb ) ( 1mg / ml ) to visualize changes in nuclear morphology . after 48 hours of treatment , stained cells were observed and photographed under a fluorescence microscope ( leica , dmls ) .
dna - fragmentation was assayed using the conventional phenol / chloroform method   and was visualized under a uv - transilluminator ( ultracam digital imaging , genei , india ) .
dna strand breakage was analysed flowcytometrically by labeling the treated and the untreated cells with 5-bromo- 2-deoxyuridine 5-triphosphate ( br - dutp )   by using bd facs verse .
the percent of cells in each phase ( subg0/g1 , g0/g1 , s , and g2/m ) was quantified flowcytometrically using bd facs verse .
rt - pcr analysis was done using primers of bax , bcl2 , cytochrome - c , caspase-3 and gapdh .
the cells were treated with ic50 dose of both drugs for 18 hours in the case of bax - bcl2 , 24 hours for cytochrome - c , and 48 hours for caspase-3 and poly ( adp - ribose ) polymerase ( parp ) .
the protein activities were measured by using indirect- enzyme linked immunosorbent assay ( elisa ) and western blot analyses .
quantifications of developed proteins after the western blot analyses were done densitometrically by using image j software .
localization of caspase-3 was done by using an immunofluorescence study   and was photographed under fluorescence microscope .
the observers were blinded during observation as to whether they were observing the control and/or drug - treated materials .
data were analyzed , and the signicance of differences between the mean values was determined by using a one - way analysis of variance ( anova ) with fisher s least significant difference ( lsd ) post hoc tests by using spss 14-software ( spss inc , chicago , il , usa ) .
ic50 values for 48 hours treatment with condurango 6c ( 3.57l/100l ) and condurango 30c ( 2.43l/100l ) against h460 were selected for the entire study ( fig 1 ) the cell viability of both placebo - treated cells at maximum dose ( 5l/100l ) was found to be very close to that of the untreated ( control ) ones .
cells were exposed to condurango 6c and 30c for 24 hours and 48 hours ( 0.5l/100l-5l/100l dose ) with respective placebos ( 5l/100l ) .
results are expressed as mean percent of cell viability  standard deviation ( sd , n = 6 ) .
signicance levels are presented as,*p < 0.05 vs. untreated cells andp < 0.05 vs. both placebo - treated cells . the light microscopy ( fig .
3 ) studies revealed that cell morphologies remained unaltered with intact cell membrane and cellular extensions in both untreated and placebo - treated cells .
however , condurango 6c - treated cells showed gradual deformation with cellular shrinkage ; condurango 30c - treated cells became gradually smaller and rounded with distorted nuclei and damaged cellular extensions , depicting the great apoptosis - inducing potential of condurango 30c .
the fluorescence intensity in optical density ( od ) was too low in untreated and placebo - treated cells at different hour intervals .
a significant increase in the fluorescence intensity was observed at 18 hours in both condurango 6c and 30c - treated cells ( fig .
fluorescence microscopy showed a greater h2 dcfda - intensity in drug - treated cells at 18 h ( fig .
signicance levels are presented as * p < 0.05 and * * * p < 0.001 between untreated and condurango 6c and 30c - treated cells and between untreated and placebo - treated cells .
results showed up - regulation and downregulation of bax and bcl2 , respectively , at 18 hours in response to both drug treatments as compared to drug - untreated cells ( fig .
condurango 30c - treated sample showed significantly increased bax and decreased bcl2-expressions than condurango 6c and placebo - treated samples .
signicance * p < 0.05 untreated ( ut ) vs condurango 6c and 30c , p < 0.05 placebo ( 6c ) vs condurango 6c andp < 0.05 placebo ( 30c ) vs condurango 30c .
figure 6a shows bright green fluorescence in drug - un - treated and cells at different time - points while condurango 6c and 30c - treated cells showed a gradual decrease in the fluorescence intensity with increasing time , but a marked decrease occurred at 24 hours , suggesting that mmp depolarizes the maximum at 24h that trigger cytochrome- c release from mitochondria .
an increased expression of cytochrome - c was found in response to drugs at 24 hour of treatment ( fig .
condurango 30c - treated cells showed more significant upregulation of cytochrome - c than condurango 6c - treated ones .
signicance levels are presented as * p < 0.05 untreated ( ut ) vs condurango 6c and 30c , p < 0.05 placebo ( 6c ) vs condurango 6c andp < 0.05 placebo ( 30c ) vs condurango 30c . a gradual increase in the dapi - fluorescence intensity
was observed in condurango 6c and 30c - treated cells at 48 hours of treatment ( fig .
further , ao / eb- staining showed both a change in the fluorescence pattern from green ( normal - cellular dna ) to orange ( nicked - cellular dna ) and an increase in the fluorescence of eb in drug - treated cells ( fig .
figure8c shows the formation of dna - laddering in both drug - treated samples , especially in condurango 30c - treated samples , compared to the dna in the drug - untreated groups .
a gradual increase in the number of dutp - nicks was noted in drug - treated cells compared to drug - untreated cells ( fig .
this strongly suggests the abilities of both drugs to generate dna - nicks that induce apoptosis .
quantitative evaluation revealed the percent of tunel - positive nuclei was greater in condurango 30c - treated cells .
the sub - diploid cell population at subg0/g1 was more highly increased in the condurango 6c and 30c - treated cells than in the placebo - treated and untreated cells ( fig .
rt - pcr and elisa data showed upregulation of caspase-3 after condurango 6c and 30c treatment as compared to untreated samples ( fig .
the western blot analysis revealed that condurango 30c had more capacity to increase caspase-3 expression significantly by forming cleaved fragments ( 20kda and 12kda ) .
p < 0.05 untreated ( ut ) vs condurango 6c and 30c , p < 0.05 placebo ( 6c ) vs condurango 6c andp < 0.05 placebo ( 30c ) vs condurango 30c .
the immunofluorescence study demonstrated a gradual increase in the localization of caspase-3 within cell - cytosol in drug - treated cells , especially in condurango 30c - treated cells ( fig .
parp activation , the end process of the caspase-3-mediated pathway is designated by the formation of two cleaved fragments of 116kda ( inactive ) and 89kda ( active ) .
the western blot analysis showed the formation of two fragments in condurango 6c and 30c - treated cells at 48 h of treatment , which was nearly absent in drug - untreated samples ( fig .
band - intensities confirmed gradual down - regulation of the 116kda fragment and significant up - regulation of the 89kda fragment , especially in the condurango 30c - treated cells .
present findings would demonstrate that exposure of nsclc- h460 cells to ic50 doses of both condurango 6c and 30c for 48 hours resulted in apoptotic cell - death .
further , the effects of condurango 30c , which actually was highly- diluted , caused relatively more palpable alterations in all parameters of this study than did condurango 6c .
this phenomenon did not apparently follow the general pharmacological rule that the effect of a drug increases linearly with its concentration , but was in line with the claim of  the higher the dilution , the stronger the effect  as per homeopathic doctrine .
incidentally , apparent evidence for discernible effects produced by two dilutions , one below and one above avogadro s limit an enigma to many rationalists who believe in the accepted laws of physical sciences , as well as pharmacological sciences .
however , claims are accumulating that homeopathic potencies beyond avogadro s limit show demonstrable beneficial/ curative effects against different diseases including cancer [ 20 - 22 ] .
in fact , our earlier in vivo findings on benzo[a ] pyrene - induced lung cancer in rats   also convincingly demonstrated the ameliorative effect of condurango 30c , for which we tried to verify that the potentized remedy showed anti - cancer potential in nsclc cells .
apoptosis induction in cancerous cells is often targeted as one of the key events of cancer chemotherapy .
thus , one approach was to look at the generation of ros and to ascertain if it had a specific role in the induction of apoptosis .
the time - course studies showed elevation of ros , mmp - depolarization and cytochrome - c release at 18 hours through 24 hours of treatment , indicating that these events were earlier than apoptotic execution at 48 hours .
our result for cytochrome - c release at 24 hours of treatment further suggested that these events occurred due to an alteration in the mitochondrial structure that could trigger apoptosis .
along with mitochondrial dysfunctions , ros generation may be initiated at early hours of drug exposure via bax- bcl2 modulation . in this study
, we found increased and decreased expression of bax and bcl2 , respectively , at 18 hours in the drug - treated series .
however , surprisingly bax - bcl2 expressions were more significant in the condurango 30c - treated samples , which is a good indication of the higher efficacy of a more highly diluted homeopathic drug . another indication of apoptosis is internucleosomal dna breakdown .
results of dapi and ao / eb - staining would suggest the formation of dna nicks , as further confirmed by dna - laddering in drug - treated cells .
tunel - positivity in both drug - treated cells as compared to placebo- treated cells would further strengthen ros - dependent dna - breakage - mediated apoptotic events .
interestingly quantitatively more dna damage was found in condurango 30c - treated cells , than was found in condurango 6c .
increased ratio of bax - bcl2 is well known to stimulate the release of cytochrome - c from mitochondria to promote activation of caspase-9 that binds to apaf-1 to lead to caspase-3 and parp - activation .
results provide clear indication of caspase-3 activation in drug - treated cells . increased expression and formation of cleavage of caspase-3 and parp after drug exposure confirmed apoptosis induction via caspase-3-mediated- intrinsic pathway .
however , noticeably , condurango 30c - treated cell showed more significant modulation of caspase-3 and parp cleavage than did condurango 6c - treated cells .
these strongly support again the idea that more potentized condurango 30c had greater ability to induce apoptosis in nsclc - h460 .
if the similar results are taken into consideration , one possible conclusion is that homeopathic drugs may act by interacting with certain high affinity receptors that regulate expressions of specific genes , but more work is necessary to identify some missing links .
the present study has gain significance because it attempts to determine if potentized forms of condurango , below ( 6c ) and above ( 30c ) avogadro s limit , which are occasionally used to treat digestive problems and stomach cancer [ 22 , 24 ] , had abilities to inhibit lung cancer progression , in vitro . the various changes noted in this study
can only be brought about by the activities of certain genes and by epigenetic modifications , which would support the hypothesis [ 3 , 23 ] that ultra - highly - diluted drugs might somehow manage to correct expressions of relevant genes , the regulation of which had failed in cancer cells .
interestingly , many factors , including the presence of nanoparticles [ 5 , 25 ] , interaction between containers ( e.g. , silica from glass or a polymer ) and drug molecules , etc .
, are now under scrutiny and are implicated in modifications of the structural orientation , the size , and the physico - chemical properties of resultant homeopathic drugs even in absence of any original molecule [ 6 , 26 ] .
the overall results of this study suggest that condurango 30c has more apoptosis - inducing ability than condurango 6c , which is consistent with the claim made in the homeopathic doctrine .
 objectives : in homeopathy , it is claimed that more homeopathically - diluted potencies render more protective / curative effects against any disease condition . 
 potentized forms of condurango are used successfully to treat digestive problems , as well as esophageal and stomach cancers . however , the comparative efficacies of condurango 6c and 30c , one diluted below and one above avogadro s limit ( lacking original drug molecule ) , respectively , have not been critically analyzed for their cell - killing ( apoptosis ) efficacy against lung cancer cells in vitro , and signalling cascades have not been studied . hence , the present study was undertaken.methods:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide ( mtt ) assays were conducted on h460-non - small - cell lung cancer ( nsclc ) cells by using a succussed ethyl alcohol vehicle ( placebo ) as a control . 
 studies on cellular morphology , cell cycle regulation , generation of reactive oxygen species ( ros ) , changes in mitochondrial membrane potential ( mmp ) , and dna - damage were made , and expressions of related signaling markers were studied . 
 the observations were done in a  blinded  manner.results:both condurango 6c and 
 30c induced apoptosis via cell cycle arrest at subg0/g1 and altered expressions of certain apoptotic markers significantly in h460 cells . 
 the drugs induced oxidative stress through ros elevation and mmp depolarization at 18 - 24 hours . 
 these events presumably activated a caspase-3-mediated signalling cascade , as evidenced by reverse transcriptase- polymerase chain reaction ( rt - pcr ) , western blot and immunofluorescence studies at a late phase ( 48 hours ) in which cells were pushed towards apoptosis.conclusion:condurango 30c had greater apoptotic effect than condurango 6c as claimed in the homeopathic doctrine . 