The In-Vitro Effect of Homeopathically Prepared Rubus idaeus and 680 nm Laser Irradiation on Cervical Cancer Cells

Abstract

Background Cervical cancer (CC) is the second leading cancer in women and is the most common in those aged 15 to 44 years. Medicinal plant extracts have been used as homeopathic preparations for health benefits. Rubus idaeus (RI) is used to treat disorders of the female genital tract and produces cytotoxic effects. However, the use of homeopathically prepared RI in combination with low level laser therapy has not previously been explored.

Aim The study aims to investigate the in-vitro effects of homeopathically prepared RI alone and in combination as a potential photosensitizer with Low-level laser irradiation (LLLI) at fluencies of 5, 10, and 15 J/cm².

Methods HeLa CC cells were treated with RI (D3, D6, and 30cH homeopathic preparations). Cells were then treated with RI IC₅₀ and 680 nm laser diode at 5, 10, and 15 J/cm² fluencies, and the results compared with untreated control cells. Trypan blue viability, lactate dehydrogenase (LDH) cytotoxicity, and adenosine triphosphate (ATP) proliferation assays were used to analyze the cellular dose-responses along with inverted microscopy, Hoechst staining and Annexin-V/PI staining.

Results RI D3 alone demonstrated an ability to reduce cellular viability to 59% and also to reduce ATP levels. The subsequent combined treatment protocol of RI D3 with all fluencies of laser demonstrated an increase in cellular ATP and increased LDH levels compared with the control.

Conclusion The increased ATP and LDH levels observed in the combined treatment protocol of 680 nm laser and RI D3 at fluencies of 5, 10 and 15 J/cm², show that the Warburg effect might have been induced in the CC cells – an increase in glucose uptake and the preferential production of lactate, even in the presence of oxygen. More research, including work on other cell lines, needs to be conducted to identify if RI and perhaps a different wavelength of laser irradiation could have potential in inducing cell death in cancer cells.

Keywords

homeopathy - photodynamic therapy - Rubus idaeus - cervical cancer - Warburg effect

• References

• 1 Meyerson M, Pellman D. Cancer genomes evolve by pulverizing single chromosomes. Cell 2011; 144: 9-10
• 2 Harden ME, Munger K. Human papillomavirus molecular biology. Mutat Res Rev Mutat Res 2017; 772: 3-12
• 3 Doorbar J. Host control of human papillomavirus infection and disease. Best Pract Res Clin Obstet Gynaecol 2018; 47: 27-41
• 4 Amaro-Filho SM, Golub JE, Nuovo GJ. et al. A comparative analysis of clinical and molecular factors with the stage of cervical cancer in a Brazilian cohort. PLoS One 2013; 8: e57810
• 5 Martin PL, Slate TA, Merritt JW. Routine smears; a practical method for cytodetection of unsuspected cervical cancer. West J Surg Obstet Gynecol 1953; 61: 102-105
• 6 Ernst E. Homeopathy: The Undiluted Facts. Including a Comprehensive A-Z. Lexicon.: Springer; 2016: 7-12
• 7 Saha SK, Roy S, Khuda-Bukhsh AR. Evidence in support of gene regulatory hypothesis: gene expression profiling manifests homeopathy effect as more than placebo. Int J High Dilution Res 2013; 12: 162-167
• 8 Jonas WB, Gaddipati JP, Rajeskhumar NV. In vitro and in vivo assessment of homeopathic treatment of prostate cancer. Paper presented at the Society of Integrative Oncology, First International Conference. New York, November 18, New York: 2004
• 9 Torres A, Vargas Y, Uribe D. et al. Pro-apoptotic and anti-angiogenic properties of the α /β-thujone fraction from Thuja occidentalis on glioblastoma cells. J Neurooncol 2016; 128: 9-19
• 10 Ferrari de Andrade L, Mozeleski B, Leck AR. et al. Inhalation therapy with M1 inhibits experimental melanoma development and metastases in mice. Homeopathy 2016; 105: 109-118
• 11 Frenkel M, Mishra BM, Sen S. et al. Cytotoxic effects of ultra-diluted remedies on breast cancer cells. Int J Oncol 2010; 36: 395-403
• 12 Giao M, Gomes S, Madureira A. Effect of in vitro digestion upon the antioxidant capacity of aqueous extracts of Agrimonia eupatoria, Rubus Idaeus, Salvia sp. and Satureja montana. Food Chem 2012; 131: 761-767
• 13 de Souza VR, Pereira PA, da Silva TL, de Oliveira Lima LC, Pio R, Queiroz F. Determination of the bioactive compounds, antioxidant activity and chemical composition of Brazilian blackberry, red raspberry, strawberry, blueberry and sweet cherry fruits. Food Chem 2014; 156: 362-368
• 14 Hsieh YS, Chu SC, Hsu LS. et al. Rubus idaeus L. reverses epithelial-to-mesenchymal transition and suppresses cell invasion and protease activities by targeting ERK1/2 and FAK pathways in human lung cancer cells. Food Chem Toxicol 2013; 62: 908-918
• 15 Chung H, Dai T, Sharma SK, Huang YY, Carroll JD, Hamblin MR. The nuts and bolts of low-level laser (light) therapy. Ann Biomed Eng 2012; 40: 516-533
• 16 Wainright M. Photodynamic therapy—from dyestuff to high-tech clinical practice. Rev Progress Coloration Related Topics 2004; 34: 95-109
• 17 Bartos A, Grondin Y, Bortoni ME. et al. Pre-conditioning with near infrared photobiomodulation reduces inflammatory cytokines and markers of oxidative stress in cochlear hair cells. J Biophotonics 2016; 9: 1125-1135
• 18 Aredes MA, Garcez MR, Chaves GV. Influence of chemoradiotherapy on nutritional status, functional capacity, quality of life and toxicity of treatment for patients with cervical cancer. Nutr Diet 2018; 75: 263-270
• 19 Dewaele M, Martinet W, Rubio N. et al. Autophagy pathways activated in response to PDT contribute to cell resistance against ROS damage. J Cell Mol Med 2011; 15: 1402-1414
• 20 Cho SH, Hong JH, Noh YW, Lee E, Lee CS, Lim YT. Raspberry-like poly(γ-glutamic acid) hydrogel particles for pH-dependent cell membrane passage and controlled cytosolic delivery of antitumor drugs. Int J Nanomedicine 2016; 11: 5621-5632
• 21 Fisher P, Leckridge B, Owen D. Principles and Practice of Homeopathy: The Therapeutic and Healing Process. London: Singing Dragon; 2015
• 22 Flores A, Sandoval-Gonzales S, Takahashi R, Krall A, Sathe L, Wei L. Increased lactate dehydrogenase activity is dispensable in squamous carcinoma cells of origin. Nat Commun 2019; 10: 91
• 23 Bensinger SJ, Christofk HR. New aspects of the Warburg effect in cancer cell biology. Semin Cell Dev Biol 2012; 23: 352-361
• 24 Doherty JR, Cleveland JL. Targeting lactate metabolism for cancer therapeutics. J Clin Invest 2013; 123: 3685-3692
• 25 Husain Z, Huang Y, Seth P, Sukhatme VP. Tumor-derived lactate modifies antitumor immune response: effect on myeloid-derived suppressor cells and NK cells. J Immunol 2013; 191: 1486-1495
• 26 Zheng X, Zhou Y, Chen W. et al. Ginsenoside 20(S)-Rg3 prevents PKM2-targeting miR-324-5p from H19 sponging to antagonize the Warburg effect in ovarian cancer cells. Cell Physiol Biochem 2018; 51: 1340-1353
• 27 Zhang R, Su J, Xue SL. et al. HPV E6/p53 mediated down-regulation of miR-34a inhibits Warburg effect through targeting LDHA in cervical cancer. Am J Cancer Res 2016; 6: 312-320
• 28 Marín-Aguilar F, Pavillard LE, Giampieri F, Bullón P, Cordero MD. Adenosine monophosphate (AMP)-activated protein kinase: a new target for nutraceutical compounds. Int J Mol Sci 2017; 18: 1025-1078
• 29 Weinberg SE, Chandel NS. Targeting mitochondria metabolism for cancer therapy. Nat Chem Biol 2015; 11: 9-15
• 30 Tyszka-Czochara M, Lasota M, Majka M. Caffeic acid and metformin inhibit invasive phenotype induced by TGF-β1 in C-4I and HTB-35/SiHa human cervical squamous carcinoma cells by acting on different molecular targets. Int J Mol Sci 2018; 19: 266