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Psorinum 6× triggers apoptosis signals in human lung cancer cells
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Research Article
Psorinum 6× triggers apoptosis signals in human lung cancer cells Jesmin Mondal1, Asmita Samadder1,2, Anisur Rahman Khuda-Bukhsh1 1. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, India 2. Department of Zoology, Dum Dum Motijheel College, Kolkata-700074, India ABSTRACT OBJECTIVE: To provide in vitro evidence of Psorinum treatment against cancer cells in a controlled study. METHODS: Effects of homeopathic Psorinum 6× on cell viability were initially determined in several cancer cell lines, including A549, HepG2 and MCF-7, using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay, and an ethanol 6× control. The cell line that exhibited highest inhibition was selected and used in the following experiments. A range of Psorinum 6× doses was used to explore treatment effects on cell cycle arrest, cell death (apoptosis), generation of reactive oxygen species (ROS) and change in mitochondrial membrane potential (MMP) using flow cytometry and fluorescence microscopy, respectively. Expression of several signal proteins related to apoptosis and cell survival were quantified with Western blotting and confocal microscopy. Further, circular dichroism (CD) spectroscopy was used to determine possible drug-DNA interactions, as well as the induction of conformational changes. RESULTS: Treatment of cancer cell lines with Psorinum showed greater anticancer effects in A549 cells than in others. In A549 cells Psorinum treatment inhibited cell proliferation at 24 h after treatment, and arrested cell cycle at sub-G1 stage. It also induced ROS generation, MMP depolarization, morphological changes and DNA damage, as well as externalization of phosphatidyl serine. Further, increases in p53 expression, Bax expression, cytochrome c release, along with reduction of Bcl-2 level and caspase-3 activation were observed after Psorinum 6× treatment, which eventually drove A549 cells towards the mitochondria-mediated caspase-3-dependent pathway. CD spectroscopy revealed direct interaction of Psorinum with DNA, using calf thymus-DNA as target. CONCLUSION: Psorinum 6× triggered apoptosis in A549 cells via both up- and down-regulations of relevant signal proteins, including p53, caspase-3, Bax and Bcl-2. Keywords: homeopathy; Psorinum therapy; lung neoplasms; reactive oxygen species; anticancer potential; apoptosis; drug-DNA interaction Citation: Mondal J, Samadder A, Khuda-Bukhsh AR. Psorinum 6× triggers apoptosis signals in human cancer cells. J Integr Med. 2016; 14(2): 143–153.
1 Introduction Great efforts have been made to successfully combat various types of cancer including improved surgical
tools, radiotherapy and chemotherapy. However, such interventions, due to financial limitations, are not available to huge populations, particularly in the developing world[1–6]. Further, the number of cancer cases
http://dx.doi.org/10.1016/S2095-4964(16)60230-3 Received June 10, 2015; accepted August 19, 2015. Correspondence: Prof. Anisur Rahman Khuda-Bukhsh; E-mail: [email protected], [email protected]
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has increased so much so that many cancer patients are unable to obtain mainstream cancer treatments because of inadequate medical infrastructure, in addition to financial constraints[5,6]. Additionally, the prognosis of some forms of cancer, like cancers of liver, gall bladder, pancreas, and stomach, is still poor[7]. Elderly cancer patients may not tolerate conventional cancer treatments because of old age-related problems[8,9], including lack of strength to cope up with chemotherapy and/or radiotherapy. Therefore, in recent years, there has been a serious search for efficient complementary and alternative medicines (CAMs) which could render additional benefits, particularly when used in combination with the mainstream Western medicines[10,11]. Psorinum therapy is one CAM practice, whose promising results have been advertised by a group of Indian scientists and clinicians[12,13]. However, these studies are based on single-armed experimental design, without any control, weakening their conclusions. Thus they do not provide the convincing evidence needed to recommend the safe use of Psorinum treatment in human subjects. In fact, in the homeopathic regimen, it has been suggested that quite a few remedies have considerable anticancer effects[14]. As a result, alternative cancer treatments have gained considerable importance in oncology throughout the world. Among several homeopathic treatment protocols now being used in India (e.g., Banerjee protocol[15] and Sankaran protocol[16]), with varying degree of success, Psorinum therapy[12,13,17,18] is believed to treat several forms of cancer successfully, enabling the patients to survive for several years with improved quality of life. The drug Psorinum 6× (“×” stands for decimal potency of homeopathy), is derived from an alcoholic extract of slough, and pus cells from scabies. The 6× potency of Psorinum is claimed to activate different immune effector cells (e.g., T cells, and accessory cells like macrophages, dendritic cells, and natural killer cells) which can trigger a complex antitumor immune response [18]. Daily oral administration of Psorinum 6× to albino rats, at doses up to 0.5 mL/kg body weight, for 2 weeks, resulted in no adverse side effects [19]. Published retrospective and prospective accounts claim considerable efficacy of Psorinum therapy in treating patients with various malignancies, namely, stomach, gall bladder, pancreatic and liver cancers, without showing any significant side effects[20]. Unless a controlled in vitro study can provide decisive proof of its anticancer potential, its human use will be restricted. Therefore the present in vitro controlled study was undertaken, using adenocarcinoma cells A549, in order to gain preliminary knowledge about its efficacy and probable molecular mechanism of action. Our focus was particularly on the signaling pathway, as cervical cancer cells HepG2 and breast cancer MCF-7 cells were also March 2016, Vol.14, No.2
found to show cytotoxicity induced by Psorinum 6× administration, in preliminary studies. 2 Materials and methods 2.1 Chemicals and reagents Dulbecco’s modified Eagle medium (DMEM), penicillin, streptomycin and neomycin (PSN) were purchased from HiMedia (India). Fetal bovine serum (FBS), trypsin and ethylene di-amine tetra-acetic acid (EDTA) were obtained from Gibco BRL (Grand Island, NY, USA). Tissue culture plastic wares were procured from Tarson (India). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), dichloro-dihydrofluorescein diacetate (H 2DCFDA), 4',6-diamidino-2phenylindole dihydrochloride (DAPI) and rhodamine 123 were purchased from Sigma (USA). Annexin V-fluorescein isothiocyanate (FITC) and primary antibodies were obtained from Santa Cruz Biotechnology Inc., (USA). Secondary antibodies were purchased from Sigma. 2.2 Source of homeopathic Psorinum 6× The Psorinum nosode is made from fluid discharged by blisters in the skin caused by a scabies (contagious mites) infestation[17]. It is considered to be one of the most effective remedies for many skin problems that do not respond to other treatments[17]. Homeopathic potencies of Psorinum 6× are made by trituration (grinding thoroughly) of the secretions with lactose, or in 70% ethanol in serial dilutions on a decimal scale (i.e., diluted in the scale of 10 at each “potentization” step of dilution). The nosode Psorinum 6× was kindly provided by Hahnemann Publishing Company Private Limited, BB Ganguly Street, Kolkata. 2.3 Cell culture MCF-7, HepG2, A549 and WRL-68 cells were collected from National Centre for Cell Science (NCCS), India. MCF-7, HepG2 and A549 cells originated from breast, liver and lung cancer, respectively. WRL68 cells were procured from NCCS Pune as certified derivatives of normal liver hepatocytes. Earlier authors have also used WRL68 as controls for normal liver hepatocytes in in vitro[21,22] experiments, as the cells could synthesize all liver marker enzymes. The cells were maintained in a humidified incubator (ESCO, Singapore) at 37 °C, with ambient oxygen and 5% carbon dioxide. Cells were cultured in DMEM with 10% heat-inactivated FBS and 1% PSN. Cells harvested with 0.025% trypsin-EDTA in phosphate-buffered saline (PBS) were plated at required cell numbers and allowed to adhere for required time before treatment. 2.4 Cell viability assay MCF-7, HepG2, A549 and WRL-68 cells were dispensed into 96-well flat bottom micro-titer plates at a density of 1×103 cells per well. Different cells were
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treated with various concentrations of Psorinum 6× (15 to 190 µg/mL) and incubated for 24 h. MTT solution (10 µmol/L) was then added to each well and incubated for 3 h at 37 °C. Formazan crystals that formed were dissolved in 100 μL acidic isopropanol and optical density (OD) was measured at 595 nm in an enzymelinked immunosorbent assay reader (Thermo Scientific, USA). 2.5 Cytotoxicity assay and selection of dose A549 cells were treated with Psorinum 6× (15 to 190 µg/mL) and MTT assay was conducted. Similarly, the MTT assay was also carried out with the normal cell lines (WRL-68) treated with Psorinum 6×. Three different doses were selected based on the MTT assay result, the 50% lethal dose (LD 50) (150 μg/mL (D3)) and two doses below the LD 50 (125 μg/mL (D2) and 100 μg/mL (D1)) for further experiments. Control cells were treated with ethanol 6× from the same stock with which the drug was prepared. 2.6 Cell morphological analysis Morphology of A549 cells treated for 24 h with the different doses of Psorinum 6× was studied along with a control group exposed to ethanol alone. A549 cells were observed under an inverted phase-contrast microscope (DMi1 Leica, Germany), equipped with a digital camera. 2.7 Nuclear morphology analysis 2.7.1 DAPI staining One control and three drug doses (100, 125 or 150 μg/mL of Psorinum 6×) were selected for this experiment. After 24-hour incubation, cells were fixed with 2% paraformaldehyde. Then the cells were stained with DAPI at 10 µmol/L concentration and observed under a fluorescence microscope (Leica DFC365 FX, Germany). 2.7.2 DNA fragmentation assay Control and drug-treated A549 cells (100, 125 or 150 μg/mL of Psorinum 6×) were washed in PBS and incubated with DNA lysis buffer (10 mmol/L Tris, 400 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, RNase (0.2 mg/mL) and proteinase K ( 0 . 1 m g / m L ) ) overnight, then centrifuged at 1 200×g at 4 °C. Supernatants were then mixed with phenol-chloroformisoamyle mixture (25:24:1) and the bi-layered mixture was centrifuged at 1 500×g at 4 °C for 15 min. DNA was then precipitated from the aqueous layer using 100% ethanol, and re-dissolved in 20 µL of Tris-EDTA buffer (10 mmol/L TrisHCl (pH 8.0) and 1 mmol/L EDTA). The extracted DNA was further purified using proteinase K and RNase A to remove contaminating proteins and RNAs, respectively. Purified DNA was separated using 1.5% agarose gel electrophoresis and bands were visualized under an ultraviolet trans-illuminator, followed by digital photography. 2.8 Drug-DNA interaction Psorinum and nuclear DNA interaction was checked Journal of Integrative Medicine
by circular dichroism spectroscopy (CD; JASCO J-720, Japan) on naked calf thymus-DNA (ct-DNA) with a drug concentration of 150 µg/mL. Placebo-treated (prepared from the same stock of ethanol that was used for “potentization” of the drug) ct-DNA was used as a control. Data were analyzed using Origin 8 Pro software (OriginLab Corporation, USA). 2.9 Cell cycle analysis PI was used for cell cycle analysis. A549 cells were treated separately with 100, 125 or 150 μg/mL Psorinum 6× for 24 h. Cells were then fixed with 70% ethanol and made RNA free. PI (5 µmol/L) was added and incubated for 20 min in the dark. Fluorescence intensity was measured using frequency lavatory-2 higher (FL-2H) with PI as the intercalating fluorescing dye. 2.10 Apoptotic analysis After treatment with Psorinum (100, 125 or 150 μg/mL), A549 cells were washed with PBS and fixed in chilled 70% ethanol. After fixation, cells were treated with RNase (5 mmol/L) and incubated for 10–15 min in the dark at 37 °C. Subsequently, cells were stained with annexin V and PI as described by Matassov et al[23]. The fluorescence intensities were determined by fluorescence-activated cell sorting (FACS) using FL-1H filter for annexin V and FL2H for PI (BD FACSCalibur, USA) to analyze apoptotic cell percentage. Data were analyzed with Cyflogic (V.1.2.1) software. 2.11 Analysis of changes in mitochondrial membrane potentials After a 24-h incubation, control and treated (100, 125 or 150 μg/mL of Psorinum 6×) cells were fixed with 2% paraformaldehyde and then incubated with 10 µmol/L rhodamine 123 for 30 min at 37 °C in the dark. After incubation, cells were immediately analyzed using florescence microscopy (Leica, Germany). 2.12 Detection of reactive oxygen species accumulation Reactive oxygen species (ROS) accumulation was assayed qualitatively after 24-h incubation with 100, 125 or 150 μg/mL of Psorinum 6×. The cells were fixed with 70% chilled ethanol and then incubated with 10 µmol/L H 2 DCFDA for 30 min at 20–25 °C in the dark. Fluorescence intensity was measured by fluorescence microscopy (Leica, Germany). 2.13 Western blot analysis A549 cells were seeded into 75-mm plates (Tarson, India) at a density of 1 × 105 cells per well. Cells were treated with different concentrations of Psorinum 6× separately and incubated for 24 h. An equal amount of protein (50 µg) was run on 12.5% sodium dodecyl sulphate polyacrylamide gel electrophoresis and electrophoretically transferred to a polyvinyl difluoride membrane. After 3% BSA blocking, the membranes were incubated overnight, at 4 °C, with specific primary antibodies, including p53,
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Bcl-2, Bax, poly (ADP-ribose) polymerase (PARP), caspase-3, cytochrome c or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The membrane was then incubated for 2 h with alkaline phosphatase (ALKP)conjugated secondary antibody. 5-Bromo-4-chloro-3indolyl-phosphate/nitro blue tetrazolium was used as developer and protein concentration was quantified by densitometry using image J software[24]. 2.14 Cytochrome c release study by confocal microscopy The cancer cells (A549) were prepared for the immunofluorescence analysis using MitoTracker Red (50 nm) and secondary fluorescent FITC antigoat antibody (Santa Cruz Biotechnology, USA). For confirmation of localization of cytochrome c in mitochondria and cytosol, control and Psorinum 6× (100, 125 or 150 μg/mL)-treated cells were incubated for 24 h with primary antibody (Santa Cruz Biotechnology, USA) at 4 oC overnight and developed with secondary FITC-conjugated anticytochrome c antibody. Then photographs were taken under a confocal microscope (Carl Zeiss LSM 510 META Laser Scanning Microscope). 2.15 Statistical analysis Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) post-hoc tests, using IBM SPSS 20 software (IBM Statistical Package for the Social Sciences Inc., 20, IBM, USA) to identify if the differences were significant among the mean-values of different groups. All experiments were done in triplicates. Results were expressed as mean ± standard error of mean (SEM). P<0.05 was considered as significant.
3 Results 3.1 Cell viability assay In the preliminary experiment, with different cancer cell lines, Psorinum 6× reduced the viability of MCF7, HepG2 and A549 cells with different doses. After 24hour incubations, 50% cell death occurred at the dose of 180.49 (±5.90), 178.27 (±2.80) and 158.24 (±6.60) μg/mL on MCF-7, HepG2 and A549 cells, respectively (Figure 1A–1B). However, the cytotoxicity was found to be significantly greater in A549 cells. The cytotoxicity was increased in A549 cells in a dose-dependent manner like that of other cell lines used. MTT results for the normal cell line (WRL-68) revealed that there was less cytotoxicity produced by Psorinum 6× after 24 h of treatment (Figure 1B). In case of WRL-68, the viability percentage was reduced by 16.27 ± 2.10 (100–83.73) at the dose of 190 μg/mL of Psorinum 6× for 24 h. 3.2 Psorinum treatment induced morphological changes in A549 cells, with chromatin condensation and nucleosomal fragmentation Morphological changes were observed in A549 cells treated with Psorinum 6×, with rounding off of the cytoplasmic periphery along with gradual detachment of cells from the substrate. Features included shrinkage of cell and blebbing of cell membrane in Psorinum-treated cells (Figure 2A). DAPI staining (Figure 2B) suggested nuclear condensation in A549 cells treated with the D3 dose of Psorinum (150 μg/mL). This result suggested that Psorinum could potentially initiate the process of cellular DNA damage by commencing with the nuclear condensation process. The results of DNA fragmentation assay further confirms the fact that Psorinum treatment
Figure 1 Cell viability assay
Initially cells (MCF-7, HepG2, A549 and WRL-68) were treated with 15–190 µg/mL of Psorinum 6× for 24 h. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results showed gradual reduction in the viability of cell lines MCF7, HepG2 (A) and A549 (B). Data are represented as percentage of control and are presented as mean ± standard error of mean. *P<0.05, vs control.
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induces cellular DNA fragmentation leading to DNA damage as observed from different fragmented bands of DNA incubated with Psorinum and compared to that of placebo-treated cells (Figure 2C). 3.3 CD spectral changes in ct-DNA by Psorinum treatment CD spectroscopic results indicated that Psorinum could interact with ct-DNA. The peak shift evident in spectra indicated that structural alterations were induced by interactions between Psorinum 6× and the DNA’s doublehelical structure (Figure 2D). 3.4 Cell-cycle arrest by Psorinum 6× on A549 cells In the cells treated with Psorinum 6×, there was a sharp increase in cells at sub-G1 (M 1) stage with respect to
the control, indicating induction of DNA fragmentation (Figure 3A). Extensive DNA damage arrests the cell at sub-G1 stage, increasing cell population at this stage. 3.5 Apoptosis analysis by FACS The measurement of apoptosis by annexin V/PI staining quantified the externalization of phosphatidyl serine. Cells showed distinct positive binding with annexin V when treated with Psorinum 6×, indicating the movement of phosphatidyl serine to the outer cell surface. Treatment with Psorinum 6× caused significant apoptotic cell death in A549 cells (Figure 3B). 3.6 Psorinum treatment depolarized mitochondrial membrane potentials The rhodamine staining assay revealed that there was
Figure 2 Psorinum treatment induced morphological changes in A549 cells
(A) Morphological analysis: A549 cells showed membrane blebbing, cell periphery shrinkage and rounding off of cells after Psorinum 6× treatment. (B) 4'-6-Diamidino-2-phenylindole dihydrochloride staining: highest chromatin condensation was observed in cells treated with 150 µg/mL Psorinum 6×, as indicated by brighter flurorescence intensity. (C) DNA fragmentation assay: results indicated that DNA-fragmentation was present in cells freated with Psorinum 6× (LN1: Control; LN2: D1; LN3: D2, LN4: D3). Fragmentation was most noticeable at the highest dose. (D) Drug-DNA interaction: circular dichroism spectra indicated ability of Psorinum 6× to interact with calf thymus DNA (ct-DNA).
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a decrease in mitochondrial membrane polarization after Psorinum 6× treatment. The reduction of mitochondrial membrane potential (MMP) was dose-dependent, and maximum depolarization was observed with 150 μg/mL of Psorinum 6× treatment (Figure 4A). 3.7 Psorinum treatment induced ROS generation The fluorescence images revealed that Psorinum treatment initiated accumulation of ROS in A549 cells. ROS generation gradually increased with an increase in
drug dose (Figure 4B). 3.8 Change in expressions of different proteins related to cytotoxicity by exposure of A549 cells to Psorinum 6× After Psorinum treatment, expression of proteins related to cell survival and apoptosis in A549 cells was quantified. The p53 expression was up-regulated by 18%, 29% and 42% with the drug doses of 100, 125 and 150 μg/mL of Psorinum 6×, respectively. Psorinum treatment caused a two-fold increase in Bax expression and an increase in
Figure 3 Data of FACS pertaining to cell cycle analysis and annexin V/propidium iodide assay
(A) Cell cycle analysis: in the drug-treated sets (100, 125 and 150 µg/mL of Psorinum 6×, respectively), there was a significant increase in cell population at M1 (=sub-G1) state of cell cycle as compared to control set, indicating the induction of DNA fragmentation after 24 h of treatment. (B) Annexin V/propidium iodide assay: A549 cells were undergoing apoptosis after Psorinum 6× treatment. Dot plot suggested that more number of apoptosis occurred at the highest dose of Psorinum 6× after 24-h incubation (Upper left=dead cells; lower left=live cells; upper right=late apoptotic cells; lower right=early apoptotic cells).
Figure 4 Fluorescence images of MMP and ROS
(A) Depolarization of mitochondrial membrane potential (MMP): fluorescence intensity was reduced gradually at increasing Psorinum doses, indicating gradual reduction of MMP. (B) Reactive oxygen species (ROS) accumulation: accumulation of ROS was linearly increased with the increase in doses (100, 125 and 150 µg/mL, respectively) of Psorinum 6×.
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total cytochrome c by 23%, 52% and 64% respectively in the A549 cell populations. This probably resulted in the release of cytochrome c and other pro-apoptotic factors from the mitochondria leading to activation of caspases. Further, PARP protein cleavage was induced by Psorinum 6× treatment in a dose-dependent manner. However, with the PARP cleavage, caspase-3 activity was also increased by 3 folds or more at the highest doses. On the other hand, both the expressions of Apaf-1 and Bcl-2 decreased upon treatment of Psorinum 6× by 24%, 29% and 30% for Apaf-1 and 2%, 12% and 28% for Bcl-2, respectively. The signaling cascade would thus indicate the induction of apoptosis in Psorinum-treated A549 cells (Figure 5A). 3.9 Cytochrome c release into cytosol Increased levels of cytochrome c in the cytosol were also shown by confocal microscopy. Experimental results indicated that cytochrome c expression was increased in the cytosolic fraction while a decrease in expression of
the mitochondrial fraction could be observed in the drugtreated group (100, 125 or 150 µg/mL), when compared with the control (Figure 5B). 4 Discussion Cancer cells are virtually immortal and characterized by uncontrolled proliferation. They do not respond to signal relating to apoptosis or cell death. Therefore, any drug or external agent gains substantial importance if it has the capacity to slow down the process of cell proliferation and trigger the signaling mechanism that initiates the process of apoptosis or cell death. The division and growth of cancer cells and their response to apoptotic factors are dependent on competition dynamics between genetically stable and unstable cells in respect of their ability to accumulate mutations[25]. In stable cells, the repair mechanism is intact, but in unstable
Figure 5 Data and images of Western blots and confocal microscopy
(A) Western blotting analysis: p53, Bax, cytochrome c, PARP and caspase-3 activities were up-regulated and Apaf-1 and Bcl-2 expressions were downregulated by Psorinum 6× treatment for 24 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was served as loading control. (B) Confocal microscopic analysis: expression and localization of cytochrome c in mitochondria and cytosol in A549 cells were studied after treatment with Psorinum 6×. Release of cytochrome c was observed in treated group as compared to control set. PARP: poly (ADP-ribose) polymerase.
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cells this mechanism does not function properly[26,27]. Mutations occur more often when DNA is damaged. There will be differential rate of accumulation of mutations in stable and unstable cells based on the presence or absence of a functioning repair mechanism. This aspect of the repair dynamics can be calculated (see Wodarz and Komarova[25] for details) and has considerable importance in ascertaining the role of carcinogens and the therapeutic value of an anticarcinogenic drug, and that drugs impact on the dynamics of development and growth of cancer cells. Most conventional chemotherapeutic drugs essentially increase the degree of DNA damage both in cancer as well as normal cells, just like a carcinogen. But in the present study, treatment with Psorinum alone did not cause any significant DNA damage in normal cells, though the DNA of cancer cells was to some extent damaged by this therapy. This can be deduced from the observed data of DAPI staining for chromatin condensation, which indicates the initiation of DNA damage. Therefore, based on the present study, it is possible that the disparity between DNA damage in normal and cancer cells may provide some therapeutic use against division and growth of cancer cells. The anticancer potential of nosodes is poorly studied[28,29], although research has shown that quite a few homeopathic remedies have anticancer potential[14]. To date, there has not been research on the effect of Psorinum 6× at the molecular level on any cancer cell line. This study is the first to apply a molecular approach towards understanding the mechanism of apoptosis by treatment with Psorinum 6× in cancer cells in vitro. Here we have presented data on the effects of three doses of Psorinum 6×, selected based on the LD50 value of the drug (158 µg/mL), with a view to identifying dose-response, if any, and to ascertain the optimum dose that shows the maximum anticancer potential of this nosode, as ascertained from data of cell viability assay. Various potencies of drug are used in homeopathy. Homeopathic potencies are prepared by a process of serial dilutions with a fixed number of jerks or succussions at each step, as described elsewhere [30]. Psorinum 6× precisely means that the initial pus from scabies, collected and fixed in ethanol (70%), was diluted with ethanol by following the succussion method of homeopathic dilutions, making the final concentration of dilution to 1 × 100 000 times. Therefore, the exact concentration of the micro-ingredients present could not be known. However, the dose-response curve (Figure 1B) looked more like one expected in accordance with general pharmacological rules of dose-response[31]. On the other hand, the “inversion effect” of homeopathic potentized drugs is commonly found when different potencies of the same drug are used on the same disease/abnormal March 2016, Vol.14, No.2
physiological state, for example, the 6C showing lesser effect than that of the more diluted 30C and 200C[32]. In this study, we selected only one potency (Psorinum 6×). Therefore, the LD50 dose as determined in cancer cells, was primarily tested for evaluating the effect of the drug, if any. Both normal and cancer cells were included to further determine if the cancer cells were affected differently than the normal control. This phenomenon of pharmacological dose-response has also been experienced by several earlier researchers, who suggested that homeopathic formulations in low to medium dilutions, containing molecules in the range of ultra-low doses, tend to exploit the extreme sensitivity of biological systems to exogenous and endogenous signals, and paradoxically show apparent pharmacological rules[31]. The indications of such medicines are determined by “proving”, i.e., by applying the remedies in healthy subjects first and observing emergence of guiding symptoms [33]. A few nosodes, derived from disease products of organs of human or animal origin[34], diluted as per homeopathic decimal/or centesimal methods, are now being used more often to treat some dreadful diseases[34,35], however, they are not considered substitutes for vaccines [36]. In lower potencies of these nosodes (e.g., 6×) there can be molecular substances like microfragments of proteins, RNA, DNA, or other components of cells out there whose effects can be felt in biological systems. The major objection to the use of homeopathic medicine is that at higher potencies at or above 12C, no original molecules of the initial drug substance can be expected to be present although nanoparticles from original drugs have been found in such ultra-highly diluted homeopathic drugs[37,38]. Further, a recent study on homeopathic dilution, carried out by a physicist [39], in the context of the Langmuir equation, inferred that homeopathic medicines may not be as dilute as a simplistic application of Avogadro’s Principle suggests, due to surface effects. Generally, there is a lesser concern shown for use of homeopathic remedies diluted below Avogadro’s limit. The 6× potency was therefore used in this study, as their original drug molecules can still be found in the drug solution, so that results could be acceptable to a greater number of patients and more confidently used by qualified homeopathic doctors for providing benefits to cancer patients, at least as in supportive cancer care. Finally, the results of this study add support to the claim that beneficial clinical results have been observed in some human cancer patients in single arm studies[13,17,40]. Drug resistance is one of the greatest difficulties for any drug in the killing of cancer cells. Therefore the resistance factor is one of the major criteria to be overcome by anticancer drugs to produce its telling effect. For this reason, to ascertain if a drug can successfully enter
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into the nucleus of the cancer cells becomes important, because proximity to the nucleus will render its effect more stronger[41]. On being satisfied that the drug entered nucleus, we became inclined to go a step further to check whether the drug in different doses could actually bind differentially with the DNA housed within the nucleus. Results of spectral band from the circular dichroism study revealed that Psorinum 6× could potentially interact with ct-DNA, changing the molecular structure and helicity of the DNA bands. The DNA fragmentation assay corroborated these findings, indicating a positive relationship between drug dose and rate of DNA damage in cancer cells. This result led us to investigate the modulation of p53 protein and cell cycle arrest of cancer cells after administration of the drug. Up-regulation of p53 protein associated with cell cycle arrest at sub-G1 stage clearly points out the actual cause of drug-induced DNA damage. The generation of ROS has been a mechanism that led to the induction of apoptosis in many anticancer drugs[42,43]. Some research suggests that cancer cells have increased level of ROS and an altered redox status[44], relative to normal cells. Therefore, it becomes necessary to speculate other factors associated with ROS. MMP is as one such phenomenon directly related to the drug-induced modulation of ROS production [45]. The present work revealed that treatment with Psorinum 6× resulted in a considerable increase in ROS generation and corresponding decrease in MMP, supporting earlier research on anticancer drugs[46]. The under-expression of apoptotic protein Bax, the over-expression of antiapoptotic protein Bcl-2 and the apoptotic assay studies with annexin V-FITC clearly indicate that there was a considerable change in the balance of these proteins’ ratio, which triggered the beginning of the apoptotic process. To further test whether the apoptotic process was activated via the intrinsic pathway, we also investigated the status of cytochrome c release, associated with the change in Bax/Bcl-2 protein ratio in drug-treated cancer cells. The images from confocal microscopy and the immunoblot assessment clearly showed that there was considerable release of cytochrome c into the cytosol from the mitochondria in drug-treated cancer cells, relative to control cancer cells. This phenomenon was further supported by caspase-3 activation indicated by the immunoblot studies. Evidence of PARP protein cleavage, leading to caspase-3 activation, corroborates other evidence that Psorinum 6× has the potential to turn on the apoptotic pathway. Overall results indicated that the drug not only interacted with the DNA double helix and induced DNA damage, but also simultaneously increased the rate of intracellular ROS, leading to the initiation of apoptosis Journal of Integrative Medicine
through the mitochondria-mediated intrinsic pathway. 5 Conclusions From the results of the present study, we can conclude that: (1) Psorinum 6× therapy has the potential to interact with the DNA and thereby to hinder the process of cell proliferation and cell cycle, the hallmark of cancerous growth; (2) the molecular mechanism involves accumulation of ROS in A549 cells and thus depolarizes MMP which initiates cytochrome c discharge into the cytosol. The actual mechanism of apoptosis also involves the up- and down-regulations of some relevant signal proteins: pro- and anti-apoptotic ones. The up-regulation of p53 can induce apoptosis in the cells by intrinsic pathways. The activated p53 in turn activates caspase-3 downstream. The alteration in the levels of Bax and Bcl-2 (Bax/Bcl-2 ratio), downstream of p53, is also a decisive factor that plays an important role in determining whether cells will undergo apoptosis leading to cell death. These events push Psorinum-treated A549 cells towards apoptosis by altering activities of different cellular proteins. 6 Acknowledgements Grateful acknowledgements are made to University of Grant Commission, New Delhi, India, for sanctioning a Junior Research Fellowship to Jesmin Mondal through Maulana Azad National Fellowship scheme and for awarding UGC Emeritus Fellowship to A.R. Khuda-Bukhsh. 7
Competing interests None to declare.
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42 Das S, Das J, Samadder A, Paul A, Khuda-Bukhsha AR. Strategic formulation of apigenin-loaded PLGA nanoparticles for intracellular trafficking, DNA targeting and improved therapeutic effects in skin melanoma in vitro. Toxicol Lett. 2013; 223(2): 124–138. 43 Trachootham D, Zhou Y, Zhang H, Demizu Y, Chen Z, Pelicano H, Chiao PJ, Achanta G, Arlinghaus RB, Liu J, Huang P. Selective killing of oncogenically transformed cells through a ROS-mediated mechanism by β-phenylethyl isothiocyanate. Cancer Cell. 2006; 10(3): 241–252. 44 Pelicano H, Carney D, Huang P. ROS stress in cancer cells and therapeutic implications. Drug Resist Updat. 2004; 7(2): 97–110. 45 Fulda S, Kroemer G. Mitochondria as therapeutic targets for the treatment of malignant disease. Antioxid Redox Signal. 2011; 15(12): 2937–2949. 46 Kuwana T, Bouchier-Hayes L, Chipuk JE, Bonzon C, Sullivan BA, Green DR, Newmeyer DD. BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Mol Cell. 2005; 17(4): 525–535.
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Research Article
Psorinum 6× triggers apoptosis signals in human lung cancer cells Jesmin Mondal1, Asmita Samadder1,2, Anisur Rahman Khuda-Bukhsh1 1. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, India 2. Department of Zoology, Dum Dum Motijheel College, Kolkata-700074, India ABSTRACT OBJECTIVE: To provide in vitro evidence of Psorinum treatment against cancer cells in a controlled study. METHODS: Effects of homeopathic Psorinum 6× on cell viability were initially determined in several cancer cell lines, including A549, HepG2 and MCF-7, using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay, and an ethanol 6× control. The cell line that exhibited highest inhibition was selected and used in the following experiments. A range of Psorinum 6× doses was used to explore treatment effects on cell cycle arrest, cell death (apoptosis), generation of reactive oxygen species (ROS) and change in mitochondrial membrane potential (MMP) using flow cytometry and fluorescence microscopy, respectively. Expression of several signal proteins related to apoptosis and cell survival were quantified with Western blotting and confocal microscopy. Further, circular dichroism (CD) spectroscopy was used to determine possible drug-DNA interactions, as well as the induction of conformational changes. RESULTS: Treatment of cancer cell lines with Psorinum showed greater anticancer effects in A549 cells than in others. In A549 cells Psorinum treatment inhibited cell proliferation at 24 h after treatment, and arrested cell cycle at sub-G1 stage. It also induced ROS generation, MMP depolarization, morphological changes and DNA damage, as well as externalization of phosphatidyl serine. Further, increases in p53 expression, Bax expression, cytochrome c release, along with reduction of Bcl-2 level and caspase-3 activation were observed after Psorinum 6× treatment, which eventually drove A549 cells towards the mitochondria-mediated caspase-3-dependent pathway. CD spectroscopy revealed direct interaction of Psorinum with DNA, using calf thymus-DNA as target. CONCLUSION: Psorinum 6× triggered apoptosis in A549 cells via both up- and down-regulations of relevant signal proteins, including p53, caspase-3, Bax and Bcl-2. Keywords: homeopathy; Psorinum therapy; lung neoplasms; reactive oxygen species; anticancer potential; apoptosis; drug-DNA interaction Citation: Mondal J, Samadder A, Khuda-Bukhsh AR. Psorinum 6× triggers apoptosis signals in human cancer cells. J Integr Med. 2016; 14(2): 143–153.
1 Introduction Great efforts have been made to successfully combat various types of cancer including improved surgical
tools, radiotherapy and chemotherapy. However, such interventions, due to financial limitations, are not available to huge populations, particularly in the developing world[1–6]. Further, the number of cancer cases
http://dx.doi.org/10.1016/S2095-4964(16)60230-3 Received June 10, 2015; accepted August 19, 2015. Correspondence: Prof. Anisur Rahman Khuda-Bukhsh; E-mail: [email protected], [email protected]
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has increased so much so that many cancer patients are unable to obtain mainstream cancer treatments because of inadequate medical infrastructure, in addition to financial constraints[5,6]. Additionally, the prognosis of some forms of cancer, like cancers of liver, gall bladder, pancreas, and stomach, is still poor[7]. Elderly cancer patients may not tolerate conventional cancer treatments because of old age-related problems[8,9], including lack of strength to cope up with chemotherapy and/or radiotherapy. Therefore, in recent years, there has been a serious search for efficient complementary and alternative medicines (CAMs) which could render additional benefits, particularly when used in combination with the mainstream Western medicines[10,11]. Psorinum therapy is one CAM practice, whose promising results have been advertised by a group of Indian scientists and clinicians[12,13]. However, these studies are based on single-armed experimental design, without any control, weakening their conclusions. Thus they do not provide the convincing evidence needed to recommend the safe use of Psorinum treatment in human subjects. In fact, in the homeopathic regimen, it has been suggested that quite a few remedies have considerable anticancer effects[14]. As a result, alternative cancer treatments have gained considerable importance in oncology throughout the world. Among several homeopathic treatment protocols now being used in India (e.g., Banerjee protocol[15] and Sankaran protocol[16]), with varying degree of success, Psorinum therapy[12,13,17,18] is believed to treat several forms of cancer successfully, enabling the patients to survive for several years with improved quality of life. The drug Psorinum 6× (“×” stands for decimal potency of homeopathy), is derived from an alcoholic extract of slough, and pus cells from scabies. The 6× potency of Psorinum is claimed to activate different immune effector cells (e.g., T cells, and accessory cells like macrophages, dendritic cells, and natural killer cells) which can trigger a complex antitumor immune response [18]. Daily oral administration of Psorinum 6× to albino rats, at doses up to 0.5 mL/kg body weight, for 2 weeks, resulted in no adverse side effects [19]. Published retrospective and prospective accounts claim considerable efficacy of Psorinum therapy in treating patients with various malignancies, namely, stomach, gall bladder, pancreatic and liver cancers, without showing any significant side effects[20]. Unless a controlled in vitro study can provide decisive proof of its anticancer potential, its human use will be restricted. Therefore the present in vitro controlled study was undertaken, using adenocarcinoma cells A549, in order to gain preliminary knowledge about its efficacy and probable molecular mechanism of action. Our focus was particularly on the signaling pathway, as cervical cancer cells HepG2 and breast cancer MCF-7 cells were also March 2016, Vol.14, No.2
found to show cytotoxicity induced by Psorinum 6× administration, in preliminary studies. 2 Materials and methods 2.1 Chemicals and reagents Dulbecco’s modified Eagle medium (DMEM), penicillin, streptomycin and neomycin (PSN) were purchased from HiMedia (India). Fetal bovine serum (FBS), trypsin and ethylene di-amine tetra-acetic acid (EDTA) were obtained from Gibco BRL (Grand Island, NY, USA). Tissue culture plastic wares were procured from Tarson (India). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), dichloro-dihydrofluorescein diacetate (H 2DCFDA), 4',6-diamidino-2phenylindole dihydrochloride (DAPI) and rhodamine 123 were purchased from Sigma (USA). Annexin V-fluorescein isothiocyanate (FITC) and primary antibodies were obtained from Santa Cruz Biotechnology Inc., (USA). Secondary antibodies were purchased from Sigma. 2.2 Source of homeopathic Psorinum 6× The Psorinum nosode is made from fluid discharged by blisters in the skin caused by a scabies (contagious mites) infestation[17]. It is considered to be one of the most effective remedies for many skin problems that do not respond to other treatments[17]. Homeopathic potencies of Psorinum 6× are made by trituration (grinding thoroughly) of the secretions with lactose, or in 70% ethanol in serial dilutions on a decimal scale (i.e., diluted in the scale of 10 at each “potentization” step of dilution). The nosode Psorinum 6× was kindly provided by Hahnemann Publishing Company Private Limited, BB Ganguly Street, Kolkata. 2.3 Cell culture MCF-7, HepG2, A549 and WRL-68 cells were collected from National Centre for Cell Science (NCCS), India. MCF-7, HepG2 and A549 cells originated from breast, liver and lung cancer, respectively. WRL68 cells were procured from NCCS Pune as certified derivatives of normal liver hepatocytes. Earlier authors have also used WRL68 as controls for normal liver hepatocytes in in vitro[21,22] experiments, as the cells could synthesize all liver marker enzymes. The cells were maintained in a humidified incubator (ESCO, Singapore) at 37 °C, with ambient oxygen and 5% carbon dioxide. Cells were cultured in DMEM with 10% heat-inactivated FBS and 1% PSN. Cells harvested with 0.025% trypsin-EDTA in phosphate-buffered saline (PBS) were plated at required cell numbers and allowed to adhere for required time before treatment. 2.4 Cell viability assay MCF-7, HepG2, A549 and WRL-68 cells were dispensed into 96-well flat bottom micro-titer plates at a density of 1×103 cells per well. Different cells were
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treated with various concentrations of Psorinum 6× (15 to 190 µg/mL) and incubated for 24 h. MTT solution (10 µmol/L) was then added to each well and incubated for 3 h at 37 °C. Formazan crystals that formed were dissolved in 100 μL acidic isopropanol and optical density (OD) was measured at 595 nm in an enzymelinked immunosorbent assay reader (Thermo Scientific, USA). 2.5 Cytotoxicity assay and selection of dose A549 cells were treated with Psorinum 6× (15 to 190 µg/mL) and MTT assay was conducted. Similarly, the MTT assay was also carried out with the normal cell lines (WRL-68) treated with Psorinum 6×. Three different doses were selected based on the MTT assay result, the 50% lethal dose (LD 50) (150 μg/mL (D3)) and two doses below the LD 50 (125 μg/mL (D2) and 100 μg/mL (D1)) for further experiments. Control cells were treated with ethanol 6× from the same stock with which the drug was prepared. 2.6 Cell morphological analysis Morphology of A549 cells treated for 24 h with the different doses of Psorinum 6× was studied along with a control group exposed to ethanol alone. A549 cells were observed under an inverted phase-contrast microscope (DMi1 Leica, Germany), equipped with a digital camera. 2.7 Nuclear morphology analysis 2.7.1 DAPI staining One control and three drug doses (100, 125 or 150 μg/mL of Psorinum 6×) were selected for this experiment. After 24-hour incubation, cells were fixed with 2% paraformaldehyde. Then the cells were stained with DAPI at 10 µmol/L concentration and observed under a fluorescence microscope (Leica DFC365 FX, Germany). 2.7.2 DNA fragmentation assay Control and drug-treated A549 cells (100, 125 or 150 μg/mL of Psorinum 6×) were washed in PBS and incubated with DNA lysis buffer (10 mmol/L Tris, 400 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, RNase (0.2 mg/mL) and proteinase K ( 0 . 1 m g / m L ) ) overnight, then centrifuged at 1 200×g at 4 °C. Supernatants were then mixed with phenol-chloroformisoamyle mixture (25:24:1) and the bi-layered mixture was centrifuged at 1 500×g at 4 °C for 15 min. DNA was then precipitated from the aqueous layer using 100% ethanol, and re-dissolved in 20 µL of Tris-EDTA buffer (10 mmol/L TrisHCl (pH 8.0) and 1 mmol/L EDTA). The extracted DNA was further purified using proteinase K and RNase A to remove contaminating proteins and RNAs, respectively. Purified DNA was separated using 1.5% agarose gel electrophoresis and bands were visualized under an ultraviolet trans-illuminator, followed by digital photography. 2.8 Drug-DNA interaction Psorinum and nuclear DNA interaction was checked Journal of Integrative Medicine
by circular dichroism spectroscopy (CD; JASCO J-720, Japan) on naked calf thymus-DNA (ct-DNA) with a drug concentration of 150 µg/mL. Placebo-treated (prepared from the same stock of ethanol that was used for “potentization” of the drug) ct-DNA was used as a control. Data were analyzed using Origin 8 Pro software (OriginLab Corporation, USA). 2.9 Cell cycle analysis PI was used for cell cycle analysis. A549 cells were treated separately with 100, 125 or 150 μg/mL Psorinum 6× for 24 h. Cells were then fixed with 70% ethanol and made RNA free. PI (5 µmol/L) was added and incubated for 20 min in the dark. Fluorescence intensity was measured using frequency lavatory-2 higher (FL-2H) with PI as the intercalating fluorescing dye. 2.10 Apoptotic analysis After treatment with Psorinum (100, 125 or 150 μg/mL), A549 cells were washed with PBS and fixed in chilled 70% ethanol. After fixation, cells were treated with RNase (5 mmol/L) and incubated for 10–15 min in the dark at 37 °C. Subsequently, cells were stained with annexin V and PI as described by Matassov et al[23]. The fluorescence intensities were determined by fluorescence-activated cell sorting (FACS) using FL-1H filter for annexin V and FL2H for PI (BD FACSCalibur, USA) to analyze apoptotic cell percentage. Data were analyzed with Cyflogic (V.1.2.1) software. 2.11 Analysis of changes in mitochondrial membrane potentials After a 24-h incubation, control and treated (100, 125 or 150 μg/mL of Psorinum 6×) cells were fixed with 2% paraformaldehyde and then incubated with 10 µmol/L rhodamine 123 for 30 min at 37 °C in the dark. After incubation, cells were immediately analyzed using florescence microscopy (Leica, Germany). 2.12 Detection of reactive oxygen species accumulation Reactive oxygen species (ROS) accumulation was assayed qualitatively after 24-h incubation with 100, 125 or 150 μg/mL of Psorinum 6×. The cells were fixed with 70% chilled ethanol and then incubated with 10 µmol/L H 2 DCFDA for 30 min at 20–25 °C in the dark. Fluorescence intensity was measured by fluorescence microscopy (Leica, Germany). 2.13 Western blot analysis A549 cells were seeded into 75-mm plates (Tarson, India) at a density of 1 × 105 cells per well. Cells were treated with different concentrations of Psorinum 6× separately and incubated for 24 h. An equal amount of protein (50 µg) was run on 12.5% sodium dodecyl sulphate polyacrylamide gel electrophoresis and electrophoretically transferred to a polyvinyl difluoride membrane. After 3% BSA blocking, the membranes were incubated overnight, at 4 °C, with specific primary antibodies, including p53,
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Bcl-2, Bax, poly (ADP-ribose) polymerase (PARP), caspase-3, cytochrome c or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The membrane was then incubated for 2 h with alkaline phosphatase (ALKP)conjugated secondary antibody. 5-Bromo-4-chloro-3indolyl-phosphate/nitro blue tetrazolium was used as developer and protein concentration was quantified by densitometry using image J software[24]. 2.14 Cytochrome c release study by confocal microscopy The cancer cells (A549) were prepared for the immunofluorescence analysis using MitoTracker Red (50 nm) and secondary fluorescent FITC antigoat antibody (Santa Cruz Biotechnology, USA). For confirmation of localization of cytochrome c in mitochondria and cytosol, control and Psorinum 6× (100, 125 or 150 μg/mL)-treated cells were incubated for 24 h with primary antibody (Santa Cruz Biotechnology, USA) at 4 oC overnight and developed with secondary FITC-conjugated anticytochrome c antibody. Then photographs were taken under a confocal microscope (Carl Zeiss LSM 510 META Laser Scanning Microscope). 2.15 Statistical analysis Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) post-hoc tests, using IBM SPSS 20 software (IBM Statistical Package for the Social Sciences Inc., 20, IBM, USA) to identify if the differences were significant among the mean-values of different groups. All experiments were done in triplicates. Results were expressed as mean ± standard error of mean (SEM). P<0.05 was considered as significant.
3 Results 3.1 Cell viability assay In the preliminary experiment, with different cancer cell lines, Psorinum 6× reduced the viability of MCF7, HepG2 and A549 cells with different doses. After 24hour incubations, 50% cell death occurred at the dose of 180.49 (±5.90), 178.27 (±2.80) and 158.24 (±6.60) μg/mL on MCF-7, HepG2 and A549 cells, respectively (Figure 1A–1B). However, the cytotoxicity was found to be significantly greater in A549 cells. The cytotoxicity was increased in A549 cells in a dose-dependent manner like that of other cell lines used. MTT results for the normal cell line (WRL-68) revealed that there was less cytotoxicity produced by Psorinum 6× after 24 h of treatment (Figure 1B). In case of WRL-68, the viability percentage was reduced by 16.27 ± 2.10 (100–83.73) at the dose of 190 μg/mL of Psorinum 6× for 24 h. 3.2 Psorinum treatment induced morphological changes in A549 cells, with chromatin condensation and nucleosomal fragmentation Morphological changes were observed in A549 cells treated with Psorinum 6×, with rounding off of the cytoplasmic periphery along with gradual detachment of cells from the substrate. Features included shrinkage of cell and blebbing of cell membrane in Psorinum-treated cells (Figure 2A). DAPI staining (Figure 2B) suggested nuclear condensation in A549 cells treated with the D3 dose of Psorinum (150 μg/mL). This result suggested that Psorinum could potentially initiate the process of cellular DNA damage by commencing with the nuclear condensation process. The results of DNA fragmentation assay further confirms the fact that Psorinum treatment
Figure 1 Cell viability assay
Initially cells (MCF-7, HepG2, A549 and WRL-68) were treated with 15–190 µg/mL of Psorinum 6× for 24 h. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results showed gradual reduction in the viability of cell lines MCF7, HepG2 (A) and A549 (B). Data are represented as percentage of control and are presented as mean ± standard error of mean. *P<0.05, vs control.
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induces cellular DNA fragmentation leading to DNA damage as observed from different fragmented bands of DNA incubated with Psorinum and compared to that of placebo-treated cells (Figure 2C). 3.3 CD spectral changes in ct-DNA by Psorinum treatment CD spectroscopic results indicated that Psorinum could interact with ct-DNA. The peak shift evident in spectra indicated that structural alterations were induced by interactions between Psorinum 6× and the DNA’s doublehelical structure (Figure 2D). 3.4 Cell-cycle arrest by Psorinum 6× on A549 cells In the cells treated with Psorinum 6×, there was a sharp increase in cells at sub-G1 (M 1) stage with respect to
the control, indicating induction of DNA fragmentation (Figure 3A). Extensive DNA damage arrests the cell at sub-G1 stage, increasing cell population at this stage. 3.5 Apoptosis analysis by FACS The measurement of apoptosis by annexin V/PI staining quantified the externalization of phosphatidyl serine. Cells showed distinct positive binding with annexin V when treated with Psorinum 6×, indicating the movement of phosphatidyl serine to the outer cell surface. Treatment with Psorinum 6× caused significant apoptotic cell death in A549 cells (Figure 3B). 3.6 Psorinum treatment depolarized mitochondrial membrane potentials The rhodamine staining assay revealed that there was
Figure 2 Psorinum treatment induced morphological changes in A549 cells
(A) Morphological analysis: A549 cells showed membrane blebbing, cell periphery shrinkage and rounding off of cells after Psorinum 6× treatment. (B) 4'-6-Diamidino-2-phenylindole dihydrochloride staining: highest chromatin condensation was observed in cells treated with 150 µg/mL Psorinum 6×, as indicated by brighter flurorescence intensity. (C) DNA fragmentation assay: results indicated that DNA-fragmentation was present in cells freated with Psorinum 6× (LN1: Control; LN2: D1; LN3: D2, LN4: D3). Fragmentation was most noticeable at the highest dose. (D) Drug-DNA interaction: circular dichroism spectra indicated ability of Psorinum 6× to interact with calf thymus DNA (ct-DNA).
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a decrease in mitochondrial membrane polarization after Psorinum 6× treatment. The reduction of mitochondrial membrane potential (MMP) was dose-dependent, and maximum depolarization was observed with 150 μg/mL of Psorinum 6× treatment (Figure 4A). 3.7 Psorinum treatment induced ROS generation The fluorescence images revealed that Psorinum treatment initiated accumulation of ROS in A549 cells. ROS generation gradually increased with an increase in
drug dose (Figure 4B). 3.8 Change in expressions of different proteins related to cytotoxicity by exposure of A549 cells to Psorinum 6× After Psorinum treatment, expression of proteins related to cell survival and apoptosis in A549 cells was quantified. The p53 expression was up-regulated by 18%, 29% and 42% with the drug doses of 100, 125 and 150 μg/mL of Psorinum 6×, respectively. Psorinum treatment caused a two-fold increase in Bax expression and an increase in
Figure 3 Data of FACS pertaining to cell cycle analysis and annexin V/propidium iodide assay
(A) Cell cycle analysis: in the drug-treated sets (100, 125 and 150 µg/mL of Psorinum 6×, respectively), there was a significant increase in cell population at M1 (=sub-G1) state of cell cycle as compared to control set, indicating the induction of DNA fragmentation after 24 h of treatment. (B) Annexin V/propidium iodide assay: A549 cells were undergoing apoptosis after Psorinum 6× treatment. Dot plot suggested that more number of apoptosis occurred at the highest dose of Psorinum 6× after 24-h incubation (Upper left=dead cells; lower left=live cells; upper right=late apoptotic cells; lower right=early apoptotic cells).
Figure 4 Fluorescence images of MMP and ROS
(A) Depolarization of mitochondrial membrane potential (MMP): fluorescence intensity was reduced gradually at increasing Psorinum doses, indicating gradual reduction of MMP. (B) Reactive oxygen species (ROS) accumulation: accumulation of ROS was linearly increased with the increase in doses (100, 125 and 150 µg/mL, respectively) of Psorinum 6×.
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total cytochrome c by 23%, 52% and 64% respectively in the A549 cell populations. This probably resulted in the release of cytochrome c and other pro-apoptotic factors from the mitochondria leading to activation of caspases. Further, PARP protein cleavage was induced by Psorinum 6× treatment in a dose-dependent manner. However, with the PARP cleavage, caspase-3 activity was also increased by 3 folds or more at the highest doses. On the other hand, both the expressions of Apaf-1 and Bcl-2 decreased upon treatment of Psorinum 6× by 24%, 29% and 30% for Apaf-1 and 2%, 12% and 28% for Bcl-2, respectively. The signaling cascade would thus indicate the induction of apoptosis in Psorinum-treated A549 cells (Figure 5A). 3.9 Cytochrome c release into cytosol Increased levels of cytochrome c in the cytosol were also shown by confocal microscopy. Experimental results indicated that cytochrome c expression was increased in the cytosolic fraction while a decrease in expression of
the mitochondrial fraction could be observed in the drugtreated group (100, 125 or 150 µg/mL), when compared with the control (Figure 5B). 4 Discussion Cancer cells are virtually immortal and characterized by uncontrolled proliferation. They do not respond to signal relating to apoptosis or cell death. Therefore, any drug or external agent gains substantial importance if it has the capacity to slow down the process of cell proliferation and trigger the signaling mechanism that initiates the process of apoptosis or cell death. The division and growth of cancer cells and their response to apoptotic factors are dependent on competition dynamics between genetically stable and unstable cells in respect of their ability to accumulate mutations[25]. In stable cells, the repair mechanism is intact, but in unstable
Figure 5 Data and images of Western blots and confocal microscopy
(A) Western blotting analysis: p53, Bax, cytochrome c, PARP and caspase-3 activities were up-regulated and Apaf-1 and Bcl-2 expressions were downregulated by Psorinum 6× treatment for 24 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was served as loading control. (B) Confocal microscopic analysis: expression and localization of cytochrome c in mitochondria and cytosol in A549 cells were studied after treatment with Psorinum 6×. Release of cytochrome c was observed in treated group as compared to control set. PARP: poly (ADP-ribose) polymerase.
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cells this mechanism does not function properly[26,27]. Mutations occur more often when DNA is damaged. There will be differential rate of accumulation of mutations in stable and unstable cells based on the presence or absence of a functioning repair mechanism. This aspect of the repair dynamics can be calculated (see Wodarz and Komarova[25] for details) and has considerable importance in ascertaining the role of carcinogens and the therapeutic value of an anticarcinogenic drug, and that drugs impact on the dynamics of development and growth of cancer cells. Most conventional chemotherapeutic drugs essentially increase the degree of DNA damage both in cancer as well as normal cells, just like a carcinogen. But in the present study, treatment with Psorinum alone did not cause any significant DNA damage in normal cells, though the DNA of cancer cells was to some extent damaged by this therapy. This can be deduced from the observed data of DAPI staining for chromatin condensation, which indicates the initiation of DNA damage. Therefore, based on the present study, it is possible that the disparity between DNA damage in normal and cancer cells may provide some therapeutic use against division and growth of cancer cells. The anticancer potential of nosodes is poorly studied[28,29], although research has shown that quite a few homeopathic remedies have anticancer potential[14]. To date, there has not been research on the effect of Psorinum 6× at the molecular level on any cancer cell line. This study is the first to apply a molecular approach towards understanding the mechanism of apoptosis by treatment with Psorinum 6× in cancer cells in vitro. Here we have presented data on the effects of three doses of Psorinum 6×, selected based on the LD50 value of the drug (158 µg/mL), with a view to identifying dose-response, if any, and to ascertain the optimum dose that shows the maximum anticancer potential of this nosode, as ascertained from data of cell viability assay. Various potencies of drug are used in homeopathy. Homeopathic potencies are prepared by a process of serial dilutions with a fixed number of jerks or succussions at each step, as described elsewhere [30]. Psorinum 6× precisely means that the initial pus from scabies, collected and fixed in ethanol (70%), was diluted with ethanol by following the succussion method of homeopathic dilutions, making the final concentration of dilution to 1 × 100 000 times. Therefore, the exact concentration of the micro-ingredients present could not be known. However, the dose-response curve (Figure 1B) looked more like one expected in accordance with general pharmacological rules of dose-response[31]. On the other hand, the “inversion effect” of homeopathic potentized drugs is commonly found when different potencies of the same drug are used on the same disease/abnormal March 2016, Vol.14, No.2
physiological state, for example, the 6C showing lesser effect than that of the more diluted 30C and 200C[32]. In this study, we selected only one potency (Psorinum 6×). Therefore, the LD50 dose as determined in cancer cells, was primarily tested for evaluating the effect of the drug, if any. Both normal and cancer cells were included to further determine if the cancer cells were affected differently than the normal control. This phenomenon of pharmacological dose-response has also been experienced by several earlier researchers, who suggested that homeopathic formulations in low to medium dilutions, containing molecules in the range of ultra-low doses, tend to exploit the extreme sensitivity of biological systems to exogenous and endogenous signals, and paradoxically show apparent pharmacological rules[31]. The indications of such medicines are determined by “proving”, i.e., by applying the remedies in healthy subjects first and observing emergence of guiding symptoms [33]. A few nosodes, derived from disease products of organs of human or animal origin[34], diluted as per homeopathic decimal/or centesimal methods, are now being used more often to treat some dreadful diseases[34,35], however, they are not considered substitutes for vaccines [36]. In lower potencies of these nosodes (e.g., 6×) there can be molecular substances like microfragments of proteins, RNA, DNA, or other components of cells out there whose effects can be felt in biological systems. The major objection to the use of homeopathic medicine is that at higher potencies at or above 12C, no original molecules of the initial drug substance can be expected to be present although nanoparticles from original drugs have been found in such ultra-highly diluted homeopathic drugs[37,38]. Further, a recent study on homeopathic dilution, carried out by a physicist [39], in the context of the Langmuir equation, inferred that homeopathic medicines may not be as dilute as a simplistic application of Avogadro’s Principle suggests, due to surface effects. Generally, there is a lesser concern shown for use of homeopathic remedies diluted below Avogadro’s limit. The 6× potency was therefore used in this study, as their original drug molecules can still be found in the drug solution, so that results could be acceptable to a greater number of patients and more confidently used by qualified homeopathic doctors for providing benefits to cancer patients, at least as in supportive cancer care. Finally, the results of this study add support to the claim that beneficial clinical results have been observed in some human cancer patients in single arm studies[13,17,40]. Drug resistance is one of the greatest difficulties for any drug in the killing of cancer cells. Therefore the resistance factor is one of the major criteria to be overcome by anticancer drugs to produce its telling effect. For this reason, to ascertain if a drug can successfully enter
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into the nucleus of the cancer cells becomes important, because proximity to the nucleus will render its effect more stronger[41]. On being satisfied that the drug entered nucleus, we became inclined to go a step further to check whether the drug in different doses could actually bind differentially with the DNA housed within the nucleus. Results of spectral band from the circular dichroism study revealed that Psorinum 6× could potentially interact with ct-DNA, changing the molecular structure and helicity of the DNA bands. The DNA fragmentation assay corroborated these findings, indicating a positive relationship between drug dose and rate of DNA damage in cancer cells. This result led us to investigate the modulation of p53 protein and cell cycle arrest of cancer cells after administration of the drug. Up-regulation of p53 protein associated with cell cycle arrest at sub-G1 stage clearly points out the actual cause of drug-induced DNA damage. The generation of ROS has been a mechanism that led to the induction of apoptosis in many anticancer drugs[42,43]. Some research suggests that cancer cells have increased level of ROS and an altered redox status[44], relative to normal cells. Therefore, it becomes necessary to speculate other factors associated with ROS. MMP is as one such phenomenon directly related to the drug-induced modulation of ROS production [45]. The present work revealed that treatment with Psorinum 6× resulted in a considerable increase in ROS generation and corresponding decrease in MMP, supporting earlier research on anticancer drugs[46]. The under-expression of apoptotic protein Bax, the over-expression of antiapoptotic protein Bcl-2 and the apoptotic assay studies with annexin V-FITC clearly indicate that there was a considerable change in the balance of these proteins’ ratio, which triggered the beginning of the apoptotic process. To further test whether the apoptotic process was activated via the intrinsic pathway, we also investigated the status of cytochrome c release, associated with the change in Bax/Bcl-2 protein ratio in drug-treated cancer cells. The images from confocal microscopy and the immunoblot assessment clearly showed that there was considerable release of cytochrome c into the cytosol from the mitochondria in drug-treated cancer cells, relative to control cancer cells. This phenomenon was further supported by caspase-3 activation indicated by the immunoblot studies. Evidence of PARP protein cleavage, leading to caspase-3 activation, corroborates other evidence that Psorinum 6× has the potential to turn on the apoptotic pathway. Overall results indicated that the drug not only interacted with the DNA double helix and induced DNA damage, but also simultaneously increased the rate of intracellular ROS, leading to the initiation of apoptosis Journal of Integrative Medicine
through the mitochondria-mediated intrinsic pathway. 5 Conclusions From the results of the present study, we can conclude that: (1) Psorinum 6× therapy has the potential to interact with the DNA and thereby to hinder the process of cell proliferation and cell cycle, the hallmark of cancerous growth; (2) the molecular mechanism involves accumulation of ROS in A549 cells and thus depolarizes MMP which initiates cytochrome c discharge into the cytosol. The actual mechanism of apoptosis also involves the up- and down-regulations of some relevant signal proteins: pro- and anti-apoptotic ones. The up-regulation of p53 can induce apoptosis in the cells by intrinsic pathways. The activated p53 in turn activates caspase-3 downstream. The alteration in the levels of Bax and Bcl-2 (Bax/Bcl-2 ratio), downstream of p53, is also a decisive factor that plays an important role in determining whether cells will undergo apoptosis leading to cell death. These events push Psorinum-treated A549 cells towards apoptosis by altering activities of different cellular proteins. 6 Acknowledgements Grateful acknowledgements are made to University of Grant Commission, New Delhi, India, for sanctioning a Junior Research Fellowship to Jesmin Mondal through Maulana Azad National Fellowship scheme and for awarding UGC Emeritus Fellowship to A.R. Khuda-Bukhsh. 7
Competing interests None to declare.
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