Homeopathy in Experimental Cancer Models: A Systematic Review

Abstract

Background Complementary and alternative medicine, including homeopathy, is widely used to improve well-being among cancer patients and reduce adverse effects of conventional treatment. In contrast, there are few studies on the use of homeopathic medicines to treat the disease itself. Yet, evidence of possible effectiveness of homeopathic high dilutions in experimental cancer models has been published during the past 20 years.

Aim The aim of the study was to perform a systematic review of fundamental research studies on homeopathic high dilutions in cancer.

Methods Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline, we conducted a literature search in the database PubMed for original publications, from 2000 to 2018 and in English, on in vitro and in vivo experimental cancer models testing homeopathic high dilutions.

Results Twenty-three articles met the inclusion criteria—14 in vitro, eight in vivo, and one in vitro plus in vivo experimental models. Most studies were from India. Research prominently focused on cytotoxic effects involving apoptotic mechanisms. Intrinsic aspects of homeopathy should be considered in experimental designs to emphasize the specificity of such effects.

Conclusion Fundamental research of homeopathy in cancer is still at an early stage and has mainly been performed by a few groups of investigators. The results point to an interference of well-selected homeopathic medicines with cell cycle and apoptotic mechanisms in cancer cells. However, these findings still need independent reproduction.

Keywords

experimental oncology - high dilutions - methodological analysis

Introduction

Cancer is the second leading cause of death worldwide, accounting for about 9.6 million deaths in 2018, and one in six deaths in general.[1]

Many cancer patients around the world have recourse to traditional, complementary, and integrative medicine to improve their well-being and check the symptoms of adverse effects of conventional therapies.[2] [3] [4] [5] [6] [7] Indeed, 8 of the 10 hospitals rated the best in 2019[8] provide such therapies, including adjuvant homeopathic treatment, with satisfactory outcomes.[9] [10] [11] [12] [13]

In contrast, there are almost no reports of homeopathic treatment of disease itself, and those available basically describe single cases or small series.[14] [15] Facing this scenario, the experience of Banerjee et al in India stands out. Their results suggest that in addition to general stimulation of the immune system, homeopathic medicines also have tumor-specific action.[16] [17] Such action, indeed, has been described in experimental studies since the early 2000s. However, to the best of our knowledge, just one single and difficult-to-access mini-review, of five pages only, has attempted a summary of all the available clinical and experimental evidence.[18] We were not able to locate any broad, encompassing review of experimental studies of homeopathic high dilutions in cancer.

Therefore, the aim of the present study was to perform a systematic review of experimental in vivo and in vitro studies of homeopathy in tumors and to identify methodological aspects that might need improvement. Based on our findings, we include some methodological recommendations for future studies.

Materials and Methods

For the present review we designed a protocol following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.[19] [20] [21] We performed a literature search in the PubMed database with the following combinations of terms: homeopathy AND cancer AND oncology AND experimental; homeopathy AND tumor AND in vitro; homeopathy AND tumor AND in vivo; homeopathy AND cancer AND in vivo; homeopathy AND cancer AND in vitro.

Eligibility criteria were: experimental in vivo or in vitro studies of homeopathic (potentized) preparations in cancer models, including different designs and controls, published in English, from 2000 to the end of 2018. We located further records through hand search of references. We considered only original studies of high dilutions exclusively; reviews, surveys, opinions or comments were excluded. We also excluded clinical studies (in both human and veterinary medicine) and experimental studies with non-tumor cells alone.

Data considered were: author(s), year, country, rationale, homeopathic medicines tested (name, dilution(s) or potency, concentration in culture medium, length of exposure), animal species/cell lines, dosage, and outcomes. We also analyzed the methodological quality of studies based on number of repetitions, randomization, blinding, and use of controls, to estimate risk of bias.

The literature search was performed by one author as per the eligibility criteria. One author jointly analyzed pre-selected titles and abstracts for inclusion. Two authors extracted the relevant data and entered them on ad hoc tables for general findings and methodological quality of in vivo and in vitro studies separately. The results were then discussed and re-checked against the original data by two authors.

Results

We initially retrieved 96 records as per the search strategy. Forty-one records were duplicates, and a further two described the same experiments as in a third study and were considered as non-eligible. Following the analysis of titles and abstracts according to the inclusion/exclusion criteria, we included 23 full-text articles for analysis—14 reporting on in vitro models, eight on in vivo models, and one on both. Thirty-two records were excluded ([Supplementary Files 1] and [2], available online only).

The results are summarized as follows: [Table 1] (in vitro) and [Table 2] (in vivo) describe general findings (author(s), year, medicines, homeopathic dilutions [named as “potencies”], concentration, length of exposure, cell line, dosage, rationale, results, and adverse events). [Table 3] describes aspects related to the methodological quality of in vitro studies (repetitions, blinding, and controls) together with a brief analysis of weaknesses and strengths, and [Table 4] the methodological quality of in vivo studies (randomization, blinding, and controls). Therefore, [Tables 3] and [4] provide a measure of risk of bias among the analyzed studies.

Table 1 General description of in vitro experimental studies

First author/year	Country	Medicines, dilution, length of exposure	Cell line	Rationale	Main findings
Şeker et al 2018[32]	Turkey	Paclitaxel, docetaxel 6x, 5c, 15c 72 h.	Human breast cancer MCF-7.	Whether standard conventional anti-breast cancer medications retain activity when in homeopathic preparation	Continuity of biological actions in high dilution: changes in gene expression, concentration-independent; disruption of microtubule structure (target of taxanes)
Munshi et al 2019[27]	India	Kojic acid, hydrogen peroxide (HP), 6-biopterin, NLE 30c 48, 96 h.	Murine B16F10 melanoma.	Changes in melanin content of melanoma cells by vitiligo-producing substances.	No cytotoxic effect. Cells treated with NLE and HP exhibited higher melanin content compared to controls at 48 h; no effect at 96 h.
Khuda-Bukhsh et al 2017[40]	India	HIV nosode, 30c 24 h.	Human lung cancer A549; WRL-68 normal liver cells (control).	Viability of tumor vs. normal cells and involved mechanisms	Cytotoxic effect; reduced viability of tumor vs. normal cells. Mechanisms: prevented cancer cell proliferation and migration, induced premature senescence, enhanced pro-apoptotic signal proteins, inhibited anti-apoptotic signal proteins, changed mitochondrial membrane potential, caused externalization of phosphatidyl serine, membrane distortion, nuclear condensation, DNA fragmentation, and ROS generation.
Joshi et al 2017[28]	India	Hydroquinone (HQ), Arsenicum sulphuratum flavum (ASF), Phosphorus 30c 48, 96 h.	Murine B16F10 melanoma	Investigation of melanogenic activity of homeopathic medicines.	No cytotoxicity. Only HQ and ASF significantly increased melanin content compared to controls and vehicle. No inhibition of tyrosinase activity.
Nascimento et al 2016[46]	Brazil	CANOVA Variable according to test.	Human lymphocytes exposed to NMU.	Investigation of antigenotoxic effects	Significant reduction of NMU-induced DNA damage and induced apoptosis.
Wani et al 2016[48]	India	Terminalia chebula MT, 3x, 6c, 30c 24 h (viability) 24 − 72 h (cell growth).	Human cancer MDA-MB-231, MCF-7, HEK.	Viability of tumor vs. normal cells.	Cytotoxic effect: decreased viability and growth of tumor cells only.
Mondal et al 2016[41]	India	Psorinum 6x 24 h	Human lung A549, liver HepG2, breast MCF-7 cancer; WRL-68 liver non-cancer cells.	Pro-apoptotic mechanisms	Pro-apoptotic effects included cell cycle arrest, reduced mitochondrial activity, increased oxidative activity, enhanced pro-apoptotic signal proteins.
Sikdar et al 2014[42]	India	Condurango 6c, 30c 24–48 hours.	Human lung cancer NCI-H460	Comparison between dilutions below and above Avogadro's number on apoptosis and involved mechanisms.	Both dilutions were effective, 30c significantly more as asserted in homeopathic theory. Mechanisms: cell cycle arrest, altered expressions of certain apoptotic markers, ROS elevation, and MMP depolarization at 18–24 h
Samadder et al 2013[43]	India	Lycopodium clavatum 5c, 15c 24 h.	Human cervical cancer HeLa; normal PBMC (control).	Anti-cancer effects of Lycopodium; comparison between dilutions below and above Avogadro's number on apoptosis and involved mechanisms.	Cytotoxic effect: decreased viability of tumor cells only. Pro-apoptotic effect: DNA fragmentation, enhanced pro-apoptotic signal proteins, down-regulation of anti-apoptotic pathways. Variable differences between the 2 tested dilutions.
Bishayee et al 2013[44]	India	Condurango 30c 48 h	Human cervical cancer HeLa.	Epigenetic modulation of histone-mediated cell cycle arrest.	Cytotoxicity: striking reduction of HDAC2 activity.
Arora et al 2013[64]	India	Sarsaparilla/human renal adenocarcinoma; ACHN + canine normal kidney cells MDCK; Ruta graveolens/colon carcinoma COLO 205; Phytolacca decandra/breast cancer MCF-7. MT, 30c, 200c, 1M, 10M 48 h.		Cytotoxicity of Banerjee protocol drugs/tumors.	Cytotoxicity and decreased proliferation of tumor cells only; greatest effect with MT, but remained in all the dilutions.
Mukherjee et al 2013[45]	India	Thuja occidentalis 30c 24 h.	BaP-intoxicated mice lung cells.	Protective role of Thuja occidentalis against normal lung cells exposed to lung carcinogen.	Increased viability of BaP intoxicated cells through down-regulation of ROS and HSP-60 and increased GSH context. No direct interaction with DNA, but striking ability to repair BaP-induced DNA damage. No effect on normal cells.
Frenkel et al 2010[49]	United States	Carcinosinum 30c, Phytolacca decandra 200c, Conium maculatum 3c, Thuja occidentalis 30c 24, 48, 72, 96 h.	Human breast cancer MCF-7, MDA-MB-231. Non-tumor human mammary epithelial cells HMLE.	Mechanism of action of Banerjee protocol drugs/tumors.	Preferential cytotoxic effects against the two breast cancer cell lines, causing cell cycle delay/arrest and apoptosis; effects were accompanied by altered expression of cell cycle regulatory proteins, and activation of the apoptotic cascade involving caspase 7 and PARP cleavage.
Wälchli et al 2006[50]	Switzerland	Cadmium chloride in potency pool (15–20c) 120 h	Human primary lymphocytes; acute T-cell leukemia (Jurkat) cells.	Hypothesis: primary cells are fitter to respond to high potencies than cell lines, especially cancer cell lines.	Increased viability of primary cells only; cancerous lymphocytes lost the ability to respond to regulatory signals and seemed unresponsive to high homeopathic potencies.

Abbreviations: BaP, benzo(a)pyrene; c, centesimal homeopathic dilution; GSH, total glutathione; HDAC, histone deacetylase; hsp, heat-shock protein; M, millesimal homeopathic dilution; MMP, mitochondrial membrane potential; MT, mother tincture; NLE, Nle4, D-Phe7]-α-melanocyte-stimulating hormone; NMU, N-methyl-N-nitrosourea; PARP, poly(ADP-ribose) polymerase; PBMC, peripheral blood mononuclear cells; ROS, reactive oxygen species; x, decimal homeopathic dilution.

Table 2 General description of in vivo experimental studies

First author/year	Country	Medicines, dilution, length of exposure	Model/rationale	Dosage	Main findings	Side effects
Andrade et al 2016[23]	Brazil	M1 homeopathic formula	Pulmonary metastatic and subcutaneous melanoma (B16F10) models/B16F10/mice C57BL Potential benefits of M1 in melanoma.	M1 inhaled (10 minutes every 12 hours) for 14 days.	Lower tumor burden in the lungs and subcutaneous tissue than control mice; tumors were impaired in proliferation and angiogenesis.	No
Banerjee et al 2010[24]	India	Chelidonium majus 30c, 200c. 30, 60, 90, 120 days.	p-DAB and PB-induced hepatocellular carcinoma/white rats (R. norvegicus). Effectiveness of Chelidonium majus to prevent carcinogen-induced hepatotoxicity.	Twice per day, gavage.	Lower incidence of tumors; reduced activity of hepatotoxicity markers and oxidative stress.	No
Kumar et al 2007[25]	India	Ruta graveolens, Hydrastis canadensis, Lycopodium clavatum, Thuja occidentalis 20c, Phosphorus 1M.	In vitro: (1) Ruta, Hydr, Lyc, Thuj on NDEA-induced hepatocellular carcinoma/Wistar rats; (2) Ruta, Phos on 3-MC-induced sarcoma/Swiss albino mice. Corroboration of previously reported anti-cancer effects.	50 µL/animal/dose, 5 days/week, gavage, concomitantly with carcinogens.	Reduced activity of hepatotoxicity markers and oxidative stress. Reduced incidence of 3-MC sarcomas and longer survival of tumor-harboring mice.	No
MacLaughlin et al 2006[22]	United States	Sabal serrulata 200c, Thuja occidentalis 1M, Conium maculatum 1M, Carcinosinum 1M. In vitro: 24–72 h In vivo: 7-day alternate drug protocol repeated over 5 weeks + 5-week post-treatment follow-up.	Preliminary in vitro experiment to select the best medicine: human prostate cancer PC-3 and DU-145; human breast cancer MDA-MB-231. In vivo experiment: PC-3, MDA-MB-231/male nude BALB/c nu + + mice Sabal specificity in prostate cancer.	Alternate drug 7-day protocol over 5 weeks, gavage.	In vitro: Sabal induced 33% decrease of PC-3 cell proliferation at 72 hours and 23% reduction of DU-145 cell proliferation at 24 h. No effect on MDA-MB-231 breast cancer cells. Thuj and Con did not have any effect. Sabal was selected for in vivo phase. In vivo: significant reduction of prostate tumor with Sabal compared to untreated controls; no effect on breast tumor growth.	Not reported
Thangapazham et al 2006[26]	United States	Conium maculatum, Sabal serrulata, Thuja occidentalis, Asterias rubens, Phytolacca decandra 30c, 200c, 1M MAT-LyLu nosode 1M.	MAT-LyLu prostate cancer/Copenhagen rats. Effect of homeopathic treatment on the expression of genes involved in apoptosis and cytokines in tumor and lung tissue and mechanisms.	100 µL/day; 7-day alternate drug protocol over 5 weeks.	Significant reduction in the tumor incidence (23%), tumor volume (45%) and tumor weight (33%). Effects not explained by changes in pro-apoptotic genes or cytokines in prostate tumor or lung metastasis.	Not reported
Pathak et al 2006[36]	India	Lycopodium clavatum 30c 7, 15, 30c, 60, 90, and 120 days.	p-DAB-induced hepatocarcinogenesis/Swiss albino mice. Test liver anti-cancer properties of Lycopodium clavatum, widely used for hepatobiliary disorders.	0.06 mL twice a day until euthanasia.	No or fewer tumors; reduced genotoxicity; reduced activity of liver toxicity markers.	Not reported
Biswas et al 2005[37]	India	Chelidonium majus and Carcinosinum 200c alone and combined 7, 15, 30, 60, 90, 120 days.	p-DAB-induced hepatocarcinogenesis/Swiss albino mice. Whether Carcinosinum may enhance the anti-cancer effect of Chelidonium majus.	0.06 mL Chel: twice a day until euthanasia; Carc: once a day until euthanasia.	One or both remedies, alone or in combination, reduced the number and size of tumors, reduced genotoxicity and activity of hepatotoxicity biomarkers.	No
Biswas & Khuda-Bukhsh 2004[38]	India	Chelidonium majus 30c, 200c.	p-DAB and/or PB-induced hepatocarcinogenesis/Swiss albino mice. Investigation of liver anti-cancer effect of Chelidonium majus, widely used for liver diseases.	0.06 mL three times/day 7 days, then twice a day until euthanasia.	Reduced number of tumors and genotoxicity; favorable modulation of toxicity marker enzymes.	No
Biswas & Khuda-Bukhsh 2002[39]	India	Chelidonium majus 30c, 200c.	p-DAB and/or PB-induced hepatocarcinogenesis/Swiss albino mice. Investigation of liver anti-cancer effect of Chelidonium majus, widely used for liver diseases.	0.06 mL three times/day 7 days, then twice a day until euthanasia.	Reduced number of tumors and genotoxicity; favorable modulation of toxicity marker enzymes; mild splenomegaly.	No

Abbreviations: 3-MC, 3-methylcholantrene; c, centesimal homeopathic dilution; M, millesimal homeopathic dilution; NDEA, N-nitrosodiethylamine; PB, phenobarbital; p-DAB, p-dimethyl amino azo benzene.

Table 3 Methodological quality of in vitro experimental studies

First author/year	Controls	Strengths	Weaknesses
Şeker et al 2018[32]	Untreated cells (negative); undiluted alcohol (named placebo); tested drugs in pharmacological concentration (positive).	Three repetitions; positive and negative controls.	No blinding; no rationale for testing diluted taxanes.
Munshi et al 2019[27]	Potentized and non-potentized alcohol.	Three repetitions	No blinding
Khuda-Bukhsh et al 2017[40]	Cells treated with potentized ethanol from the same stock.	All experiments were done in triplicate and replicated thrice; multiple experiments on same model.	Blinding not reported; no rationale for action of HIV nosode in lung cancer.
Joshi et al 2017[28]	Potentized and non-potentized alcohol.		No repetitions; blinding not reported.
Nascimento et al 2016[46]	Non-exposed untreated cells (negative); exposed untreated cells (positive); non-exposed treated cells; exposed treated cells.		Blinding not reported; no repetitions.
Wani et al 2016[48]	40 to 5% alcohol (pre-test); untreated cells (negative). The dilutions of alcohol to test its toxicity (pre-test) were 1: 2.5 up to 1:20.	Compared cancer and non-cancer cells; alcohol toxicity in different concentrations; toxicity investigated in pre-test; nanoparticle analysis.	No repetitions; no blinding.
Mondal et al 2016[41]	Alcohol 6x	Choice of sensitive cell line in pilot study; several assays to elucidate mechanisms.	No repetitions; no blinding; rationale based only on clinical empirical data.
Sikdar et al 2014[42]	Vehicle	Blinding; several analysis methods including key proteins in apoptosis and cell morphology.	No repetitions
Samadder et al 2013[43]	Untreated HeLa cells (negative); treatment with vehicle (placebo, positive); conventional chemotherapy agent (positive); PBMC (comparison).	Comparison of cancer and non-cancer cells; positive, negative, and comparison controls.	No repetitions; no blinding.
Bishayee et al 2013[44]	Non-potentized alcohol.	Blinding	No repetitions
Arora et al 2013[64]	Untreated cells; vehicle.	Comparison of cancer and non-cancer cells.	No repetitions; no blinding.
Mukherjee et al 2013[45]	Untreated cells (positive); vehicle (negative).	Three repetitions; positive and negative controls; simultaneous analysis of several parameters.	No blinding
Frenkel et al 2010[49]	Untreated cells; vehicle.	Treatments at different times and different cells lines; simultaneous analysis of several apoptosis and oncogenesis aspects.	No repetitions; no blinding.
Wälchli et al 2006[50]	Pool de-ionized water potencies (15–20c).	Partial blinding; cells challenged with several concentrations of the toxic agent.	No repetitions; one single parameter of analysis.

Abbreviations: Potentized, submitted to serial dilution and agitation following homeopathic technique; PBMC, peripheral blood mononuclear cells.

Table 4 Methodological quality of in vivo experimental studies

First author/year	Randomization	Blinding	Controls
Andrade et al 2016[23]	Not reported	Yes	Animals treated with vehicle.
Banerjee et al 2010[24]	Yes	Untreated animals (negative); potentized alcohol; animals treated with p-DAB (positive); animals treated with p-DAB and potentized alcohol.
Kumar et al 2007[25]	Not reported	No	Potentized alcohol; untreated, unchallenged animals (healthy).
MacLaughlin et al 2006[22]	Not reported	No	Untreated, unchallenged animals; potentized water.
Thangapazham et al 2006[26]	Not reported	No	Animals treated with potentized water.
Pathak et al 2006[36]	Not reported	No	Unexposed untreated; unexposed treated with potentized vehicle; exposed untreated; exposed treated with potentized vehicle; exposed treated with Lyc.
Biswas et al 2005[37]	Not reported	No	Animals treated with vehicle.
Biswas & Khuda-Bukhsh 2004[38]	Not reported	No	Animals treated with vehicle.
Biswas & Khuda-Bukhsh 2002[39]	Not reported	No	Untreated animals.

Abbreviation: DAB, dimethyl amino azo benzene.

One study[22] included both in vitro and in vivo models, the in vitro step having been performed to select the best medicine to be used in vivo. Since its main results concern the latter stage, we describe it together with the other in vivo studies. Interestingly, the results of both stages were convergent.

Overall, our analysis indicates that little experimental research has been done on homeopathy in cancer, and without any significant temporal trend. The largest proportion of studies was performed in India (13 of 22 studies, 63.6%), with a few contributions from the United States (n = 3), Brazil (n = 2), Switzerland (n = 1), and Turkey (n = 1). In addition, all three studies performed in the United States have Indian co-authors, and 10 of the 13 studies conducted in India were chaired by Khuda-Bukhsh.

Models, protocols and selected parameters follow the authors' research program/interests; thus the findings exhibit substantial heterogeneity that hinders drawing broad-scoped inferences. For this reason, in the next section we discuss the studies according to their group affiliation.

Several studies investigated direct anti-tumorigenic effects in vivo together with their possible mechanisms, including inhibition of cell proliferation, angiogenesis,[23] oxidative stress,[24] [25] and gene and cytokine regulation—in the latter case, ruled out.[26]

In regard to methodological aspects, most authors did not report whether the studies were blinded or not—the same was the case for randomization among the in vivo studies. A large proportion of in vitro experiments comprised a single experiment, i.e., without any repetition. Controls varied considerably among the studies: most did not include negative and positive controls, but some included a comparator.

Finally, the two studies by Munshi et al[27] [28] do not strictly address homeopathy in cancer, but used melanoma cells in the attempt to understand the mechanisms underlying homeopathic treatment of vitiligo. For this reason, we do not discuss them further here.

Discussion

A total of 23 records—12 in vitro, eight in vivo, and one with both in vitro and in vivo experimental models—met the inclusion/exclusion criteria and were considered for analysis. Such small number might be due to the rigorous eligibility criteria we established to ensure that only high-quality, peer-reviewed, and widely accessible studies would be considered for analysis.

The leading position of India in homeopathy research on cancer since the 2000s was evident also in this study and makes further comments unnecessary.[29] [30]

The best effect on tumor reduction in vitro was obtained on prostate tumor cells with repeated administration of Sabal serrulata, which was confirmed in vivo.[22] In turn, Andrade et al described an innovative in vivo protocol[23] in which mice were continually exposed to a homeopathic complex (M1) for 14 days through a nebulizer, resulting in significant reduction of transplanted B16-F10 melanoma cells. These two studies point to a relevant issue to be considered in future experimental designs: in what measure are repeated doses necessary to achieve significant effects on tumor development? In addition, we should observe that Oliveira et al recently reproduced the same effects obtained by Andrade et al[23] in an in vitro model, adding a mechanistic molecular explanation to the observed effects in single B16-F10 cell cultures.[31]

In their study, Şeker et al[32] sought to identify continuity between the effect of taxanes as used in conventional treatment for breast cancer and the same drugs in high dilution. Besides a demonstration of effectiveness of high dilutions, it is very difficult to infer any application for this type of research, since effects were less significant with high dilutions compared to pharmacological dosage. In addition, the authors do not seem to have designed their study to verify some of the known features of high dilutions, such as linearity, non-linearity, or inversion of high dilution effects[33] [34] [35]—which might be an interesting contribution of further studies—since these topics are not considered in their discussion.

The group chaired by Khuda-Bukhsh has developed a consistent line of research during the analyzed 20 years. Their earliest work corresponds to in vivo studies to investigate liver anti-cancer effects of homeopathic medicines that have been long known in clinical practice for liver conditions, namely Chelidonium majus and Lycopodium clavatum. For this series of studies, these authors had recourse to a single model—p-DAB and PB-induced hepatocarcinogenesis in Swiss albino mice, and analyzed number and size of tumors, genotoxic potential, and activity of liver enzyme biomarkers, with significant effects among the treated animals compared to controls.[36] [37] [38] [39] Next they sought to establish whether Carcinosinum, i.e., the cancer nosode, could enhance these anti-cancer effects.[37]

The studies performed by this group in the 2010s had a new and different focus: to document pro-apoptotic effects of homeopathic medicines on cancer cells and establish their underlying mechanisms.[40] [41] [42] [43] [44] [45] In all these cases, they reported positive findings—cytotoxic action, involving several signal proteins, the mitochondrial membrane potential, oxidative activity, cell cycle arrest, and DNA damage. These findings agree with those reported by Nascimento et al[46] for CANOVA—a formula that combines the homeopathic medicines Aconitum napellus, Arsenicum album, Bryonia alba, Lachesis muta and Thuja occidentalis in dilutions 11x to 19 x .[47]

The hypothesis that homeopathic medicines only hinder the viability of tumor cells, without any cytotoxic activity on normal ones, was tested by Wani et al,[48] and Frenkel et al,[49] both of which groups analyzed drugs and cancer models included in the Banerjee protocol. Against this evidence, however, Wälchli et al[50] hypothesized and demonstrated that primary cells are fitter to respond to high potencies than cell lines, especially cancer cell lines. The alleged reason is that cancer cells lose the ability to respond to regulatory signals, and thus might become unresponsive to subtle stimuli such as those represented by homeopathic high dilutions.

The study by MacLaughlin et al[22] stands out as regards anti-tumorigenic effects in vivo. Sabal serrulata (Serenoa repens) has long been known for its effects on benign prostatic hyperplasia, both in pharmacological and in homeopathic doses.[34] [47] These authors sought to establish whether this medicine has, indeed, preferential action in the prostate. Therefore, they tested its effects on prostate and breast cancer in vivo and in vitro and compared the effects on the former to other homeopathic medicines widely indicated for tumors in general (Conium maculatum, Thuja occidentalis, Carcinosinum). The results confirmed their hypothesis and are even more robust since there was agreement between the in vitro and in vivo findings.

The variability we found in selected controls among the analyzed studies points to a very serious issue in high dilution research: namely, what comprises ideal controls. In principle, positive and negative controls are assumed to be the best parameters to establish the presence and degree of a certain effect: for instance, cytotoxicity against tumor cells in vitro. However, some variables are critical in the case of high dilution research, even though they are irrelevant in conventional pharmacological studies. For example, using non-succussed water or untreated cells may hide eventual non-specific effects resulting from succussion, such as leaching from flask walls[51] and generation of nanobubbles,[52] which might change several physico-chemical parameters of water or other vehicles.[53] [54] [55] This point is critical for in vitro but less relevant for in vivo studies, due to the complex interaction of high dilutions with gastrointestinal tissues. In some cases, this interaction is not even needed.[23] In turn, blinding and randomization of animals are fundamental methodological criteria in vivo. Researchers and animals systematically interact during experimental procedures, which may act as a potential cause of bias. As [Table 4] shows, only one of nine studies reported randomization and two of nine reported blinding.

Again, as concerns controls, the use of succussed alcohol is a subject of controversy. Diluted and agitated alcohol is no mere “potentized vehicle”, but a proper homeopathic medicine—Alcoholus or Ethylicum—with its specific set of signs and symptoms obtained in homeopathic pathogenetic trials.[56] In addition, some authors reported specific effects of potentized alcohol in experimental models in vitro by comparison to other controls.[57] [58] Therefore, potentized alcohol seems to be more of a comparator than a negative control. Non-potentized alcohol may be used instead, or together with succussed water. A recent experimental in vitro study showed how important is it to characterize non-specific effects of vehicles.[59] From this perspective, using both succussed and non-succussed vehicles may represent a good standard for controls in future studies in vitro. As shown in [Table 3], only two studies used both types[27] [28] whilst most only used vehicle and untreated cells, following the standards of conventional pharmacological research.

The choice of medicines and potencies, as well as their relationship to the main effect, is still unclear. In addition, the possibility of improving outcomes through a combination of potencies (known as “potency chord”)[60] is still an open field for further investigation. One single study implemented this experimental design.[50]

Based on our findings, we offer the following recommendations for future studies:

1. Rationale: Investigators should have a clear idea of what they intend to demonstrate in each study and what implications are in terms of biological and pharmacological theory. As is universally known, fundamental research usually does not generate findings with immediate practical applications. Therefore, investigators should not assume that their results will in any way contribute to the actual treatment of cancer patients, human or animal, although this might be the main motivation for studies. No in vitro study can be used to validate empirical uses of some medicines, considering that the tissue micro-environment is a crucial factor in tumor progression.[60] In vivo studies and clinical trials are mandatory to validate new therapies, provided they systematically comply with ethical standards. Citations used to ground experiments should be duly screened and checked for reliability. When reporting conclusions, investigators should clearly state what the implications of their study are for the state of the art and how it may contribute to future research.

2. Blinding and randomization: Both are essential requirements in scientific research of homeopathic high dilutions to reduce bias, since these medicines’ mechanism of action is still unknown, and many unexpected variables may interfere somehow with the results. These two procedures,[61] plus allocation concealment, should be systematically reported.

3. Statistical analysis: All care should be taken to include the due statistical analysis of results. In many of the analyzed studies, for instance, there was no indication of the statistical significance of findings (e.g., number of tumors between treated and untreated groups).

4. Controls: All studies, without any exception, should include positive and negative controls, which should be clearly described in a separate sub-section. Study designs with different vehicle conditions (succussed water and non-succussed alcohol) must be considered since the agitation seems to be crucial to reveal any effect of homeopathic medicines. However, it can also produce several physical changes in water or other solvents likely to induce non-specific effects.[59]

5. Alcohol: Alcohol is not at inert substance in either pharmacological dose or homeopathic preparation.[62] As in the case of the former, any effect of potentized alcohol cannot be considered a non-specific “vehicle effect”, but a specific effect of the homeopathic medicine Alcoholus/Ethylicum. In addition, all studies on cells should include a pre-test of alcohol cytotoxicity or a specific citation on this issue. We further suggest preparing final working potencies in sterile water from conventional hydro-alcoholic solutions.[63]

6. Repetitions: All in vitro experiments yield more reliable outcomes when they are repeated and average results of independent series of tests are reported. Even better, they should be reproduced by different laboratories, or tested in multi-center studies.

Conclusion

Fundamental research of homeopathy in cancer is still at an early stage and mainly performed by single groups of investigators, mostly from India. Results should be reproduced at different laboratories and also in other countries.

Our results point to an interference of homeopathic high dilutions with the cell cycle and apoptotic mechanisms in cancer cells. The available evidence suggests that well-selected homeopathic medicines have preferential cytotoxic action on cancer versus normal cells.

One of the analyzed studies had a broad encompassing design, based on a solid clinical background, and with coherent results observed in both in-vivo and in-vitro experiments: it may serve as a model for further initiatives. Additional methodological refinement based on specific aspects of high dilutions is necessary, such as the choice of the best controls.

Conflict of Interest

None declared.

Acknowledgments/Funding

The authors would like to thank Universidade Paulista for the PhD grants to Andreza Pereira dos Santos and Thayná Neves Cardoso. The study had no other funding source.

Highlights

• Effectiveness of homeopathic products on cancer has been experimentally tested since the early 2000s.

• A systematic review of these studies was performed using PRISMA methods.

• Cell cycle arrest and increase in apoptosis rate were the most reported findings.

• Methodological deficiencies were revealed among the studies.

• Recommendations for further studies are suggested.

• References

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Publication History

Received: 13 April 2020

Accepted: 15 June 2020

Article published online:
21 December 2020