r e s e a r c h w o r k Samrat Ghosh Department of Zoology University of Kalyani

Transcription

1 Assessment of anticancer potentials of certain homeopathic mother tinctures, their biologically active principal ingredients and potentized forms on human cancer cell lines, A375 and HepG2 Samrat Ghosh Department of Zoology University of Kalyani S u m m a r y o f r e s e a r c h w o r k

2 Assessment of anticancer potentials of certain homeopathic mother tinctures, their biologically active principal ingredients and potentized forms on human cancer cell lines, A375 and HepG2 of research work Submitted for partial fulfillment of the Degree of Doctor of Philosophy (Science) University of Kalyani 2013 Name of candidate: Samrat Ghosh Reg. No. - Ph.D/Regn./N.Rgl./Sc 94/Zool./SG/2011 Department of Zoology University of Kalyani.

3 Introduction: In course of human evolution, plants played a great role providing not only food, but the first medicine was also probably provided by them. Traditional plant based medicines still exert a great deal of importance. Some of these so called medicinal plants have subsequently been researched for scientific validation and some led to discovery of drug candidates for combating a variety of critical diseases that threaten human health. Various parts of the medicinal plant like roots, stem, leaves, flowers or spores or pollens have been used for extracting materials in either water or alcoholic or any other form of non-toxic solvents. These plant extracts have long been used as herbal medicine or as part of ingredient of other form of traditional medicine (TM), and occasionally also in orthodox medicines. Alcoholic plant extracts have mostly been utilized as remedies for various ailments by several forms of traditional medicines like Ayurveda, Unani etc and in complementary and alternative medicines (CAM) like homeopathy. As death toll from some infectious diseases has declined in this world, cancer has become the leading cause of death now. Cancer affects people of all ages, but nearly all types of cancer are more common in middle aged and elderly people than in young. In 2010, it accounted for nearly 13% of all deaths worldwide (WHO). Cancer develops gradually as a result of a complex mix of factors, related to environment, lifestyle and heredity such as tobacco consumption, UV-exposure, radiation, arsenic poisoning etc. Actually, cancer is the name for disease in which group of cells become abnormal (no longer respond to normal growth control mechanisms) and display uncontrolled growth and invasion. They may spread through the blood stream and lymphatic system to other parts of the body. Skin cancers are skin growths with differing causes and varying degrees of malignancy. Skin cancer generally develops in the epidermis (the outermost layer of skin), so a tumor can usually be seen. The three most common malignant skin cancers are basal cell cancer, squamous cell cancer, and melanoma, each of which is named after the type of skin cell from which it arises. Skin melanoma is the most common malignancy arising in the post transplantation setting. Skin melanoma is associated with rapid proliferation, numerous early metastases and high resistance to conventional treatment. Preventive measures like chemotherapy are useful but have chronic side-effects too. Skin cancer is often metastasized to the organs like lung, liver, bone, brain, adrenal glands, gastrointestinal tract etc. and forms secondary cancers.

4 Liver cancer, formed also from metastasized malignant melanoma, is another common cancer in the world. There are many forms of liver cancer, although many cancers found in the liver are secondary invasions (metastases from other tumors), the most frequently encountered liver cancer is hepatocellular carcinoma (HCC) (also named hepatoma, which is a misnomer because adenomas are usually benign). This tumor also has a variant type that consists of both HCC and cholangio-carcinoma components. Treatment of these deadly forms of cancers generally includes surgery, chemotherapy, and radiotherapy. Although preventive measures like chemotherapy are very useful but it has some chronic side-effects too, like nausea and vomiting, muscle and nerve problems, anemia and low red blood cell count, hair loss, fatigue, diarrhea, shortness of breath, mouth sores and damaged veins. Moreover the orthodox types of treatments are very costly and often become almost impossible to bear the expense for the people living under poverty line. And unfortunately, a huge number of people in countries like India, and certain other countries live at or below poverty line, necessitating availability of effective drugs at an affordable cost. India is a big resource of biodiversity including many herbs with great medicinal implications and many homeopathic drugs are derived from these plants. Another feature that makes homeopathy, an alternative mode of treatment, to be a subject of exploration in recent years is that it has no such known side effects and homeopathic drugs are quite cheap in price and easily available in the market. In this scenario, homeopathic drugs (alone or in combination with synthetic therapeutic agents) may be useful for people suffering with a fatal disease like cancer, particularly if the anti-cancer potentials of the homeopathy drugs can be scientifically established. Incidentally, research works on anticancer potentials of some homeopathic drugs (such as Thuja Occidentalis, Lycopodium Clavatum) have already been carried out and found to have considerable potentials. Objectives: With this background scenario, the main objectives of the present investigation are: Assessment of anticancer potentials of ethanolic extract of Phytolacca decandra (homeopathic mother tincture) and its biologically active principal ingredient, oleanolic acid on human skin melanoma (A375) cells and human hepatocellular carcinoma (HepG2) cells

5 Assessment of anticancer potentials of ethanolic extract of Ruta graveolens (homeopathic mother tincture), its biologically active principal ingredient (graveoline) and potentized forms (Ruta 6C and Ruta 30C) on human skin melanoma (A375) cells and on human hepatocellular carcinoma (HepG2) cells The total work has been divided into two chapters on the basis of cancer types, namely, skin cancer and liver cancer, against which the anticancer potential of the homeopathic drugs also of their active principal ingredient has been evaluated. Both the chapters have again been subdivided into five parts each that would deal with different aspects of the study. Chapter 1: Assessment of anticancer potentials of ethanolic extract of Phytolacca decandra and Ruta graveolens, their biologically active principal ingredients oleanolic acid and graveoline, and potentized forms of Ruta graveolens on human skin melanoma (A375) cells. The work of the chapter 1 has been described under the following 5 sub-sections: 1. Anticancer effect of ethanolic extract of Phytolacca decandra (PD) on skin melanoma (A375) cells. 2. Anticancer effect of oleanolic acid (biologically active ingredient of Phytolacca decandra) on skin melanoma (A375) cells. 3. Anticancer effect of ethanolic extract of Ruta graveolens (RG) on skin melanoma (A375) cells and against DMBA induced skin cancer in Swiss albino mice in vivo. 4. Anticancer effect of graveoline (biologically active ingredient of Ruta graveolens) on skin melanoma (A375) cells. 5. Anticancer effect of potentized forms of Ruta graveolens (Ruta 6C and Ruta 30C) on skin melanoma (A375) cells. Chapter 2: Assessment of anticancer potentials of ethanolic extract of Phytolacca decandra and Ruta graveolens, their biologically active principal ingredients oleanolic acid and graveoline, and potentized forms of Ruta graveolens on human hepatocellular carcinoma (HepG2) cells. The work of the chapter 2 has been described under the following 5 sub-sections: 1. Anticancer effect of ethanolic extract of Phytolacca decandra (PD) on hepatocellular carcinoma (HepG2) cells.

6 2. Anticancer effect of oleanolic acid (biologically active ingredient of Phytolacca decandra) on hepatocellular carcinoma (HepG2) cells. 3. Anticancer effect of ethanolic extract of Ruta graveolens (RG) on hepatocellular carcinoma (HepG2) cells. 4. Anticancer effect of graveoline (biologically active ingredient of Ruta graveolens) on hepatocellular carcinoma (HepG2) cells. 5. Anticancer effect of potentized forms of Ruta graveolens (Ruta 6C and Ruta 30C) on hepatocellular carcinoma (HepG2) cells. Methods applied Before examining these aspects a detailed literature study has been presented on the related subject matters and those are mentioned in the Literature review section. Several parameters like mass spectroscopy, 1H NMR, FTIR, cell viability Assay, CD spectroscopy, thermal denaturation study, LDH based cytotoxicity assay, morphological analysis by phase contrast microscopy, DNA fragmentation assay, monolayer growth assay, soft agar colony forming assay, migration assay, fluorescence microscopic analysis with AO/EB staining, DAPI staining, H2DCFDA staining, Rhodamine 123 staining, acridine orange staining, Hoechst staining, detection of localization of some relevant signalling proteins by confocal microscopy, immunofluorescence analysis, flow cytometric analysis for various aspects like quantification of ROS accumulation, mitochondrial membrane depolarization, acidic vesicular organelles (AVOs), cell cycle progression, annexin V-FITC/PI dual stain apoptosis assay, expression study of some genes by indirect staining method, expression study of some relevant signaling genes by semi-quantitative RT-PCR analysis, ELISA assay and western blot analysis etc have been performed by deploying the standard protocols. For in vivo experiments tumor incidence, tumor volume, tumor burden, biochemical estimation of various enzymes like LPO, GSH, GR, SOD, CAT, GP x GST and GR have been done. To examine several protein level expressions ELISA and immunoblot analyses were done. Statistical analysis was performed by one-way ANOVA with post-hoc LSD tests, using SPSS 14 software to identify if the differences were significant among the mean-values of different groups. Results were expressed as Mean±SE (Standard Error).The *p<0.05 was considered to be significant. Results and Discussions:

7 Overall results indicated that ethanolic extract of Phytolacca decandra (PD) was not cytotoxic to normal PBMC and normal liver cells, but showed profound anticancer effect on skin melanoma cells by indulging the cells to apoptosis via elevation of ROS. This study also indicated that ROS might activate p53 to induce apoptosis via caspase 3 mediated pathway. On the other hand PD showed anti-liver cancer effect by inducing cell death through ROS mediated inactivation of survival pathways like NFκβ and Akt in HepG2 cells. Similarly, overall results suggested that Oleanolic acid (OA), a successfully isolated saponin from ethanolic extract of Phytolacca decandra by us, was non-toxic to normal cells and at an early hour, it targeted nuclear DNA of HepG2 cells, reduced proliferation by targeting epidermal growth factor receptor and selectively triggered apoptotic cell death in HepG2 cells through a mitochondria dependent caspase mediated pathway. OA produced similar results of inducing apoptosis preceded by interaction with nuclear DNA when administered to skin melanoma cells. At an early hour its interaction with nuclear DNA would give it an added advantage over other drugs, in producing the desired anti-cancer effects. Ethanolic extract of Ruta graveolens (RG) was non-toxic to normal cells, reduced proliferation and triggered autophagic and mitochondria dependent caspase mediated apoptotic cell death of both A375 and HepG2 cells. To ascertain if RG had bearable amount of toxicity and yet effective on in vivo system, mice (Mus musculus) were subjected to topical application of DMBA, a carcinogen, to induce skin cancer and administered RG to find out if it could render any protective effects in mice. In the present study, RG administration (75 mg/kg body weight) neither showed any acute nor chronic toxicity in mice, but showed significant reduction in the skin tumor burden in DMBA painted mice. Mice only treated with DMBA showed 100% tumor incidence whereas oral administration of RG alongside strikingly prevented the formation of well differentiated skin carcinoma in DMBA treated animals; RG possibly acted through activation of multiple biochemical mechanisms including phase-ii detoxification enzyme induction and antioxidant defence mechanism. Findings of the present study also indicated that RG had a potent antilipid peroxidative and antioxidant function during DMBA-induced skin carcinogenesis. The present study thus demonstrated the chemo preventive potential of RG in DMBA-induced skin carcinogenesis. Graveoline; successfully isolated alkaloid from ethanolic extract of Ruta graveolens by us induced ROS mediated autophagic and apoptotic cell deaths in both skin melanoma and

8 hepatocellular carcinoma cells and that autophagic cell death induction was Beclin-1 associated and independent of apoptosis; these provide a novel information that can help in newer cancer drug design, particularly in view of the fact that cancer cells sometimes become able to block the induction of apoptosis resulting in drug resistance. This may help develop new strategies for formulation of effective therapeutic drugs that will selectively target cancer cells to undergo autophagy induced cell death independent of the normal apoptotic pathway that is generally targeted. Both the potentized forms of Ruta graveolens, namely, Ruta 6C (R6) and Ruta 30 C (R30) showed profound anticancer effect on both A375 and HepG2 cells by pushing the cells towards caspase 3 mediated apoptosis via elevation of ROS. Therefore, results of the present study would validate the efficacy of ultra-highly diluted and potentized Ruta 6C and Ruta 30C to also have considerable anti-cancer potential, which might encourage their use by the CAM practitioners at least as supportive medicines in cancer therapeutics. Present study has clearly demonstrated evidences that the homeopathic drugs, namely, Phytolacca decandra and Ruta graveolens, their bio-active principal ingradients (oleanolic acid and graveoline, respectively) and potentized forms (Ruta 6C and 30C) had antiproliferative, anti-cancer activities by modulating several proteins expression and ultimately inducing programmed cell death in both skin melanoma and hepatocellular carcinoma cells, in vitro, suggesting thereby their usefulness as anti-cancer drugs against both skin cancer and liver cancer, opening up alternative possibilities of combined therapies in increasing survivability of skin and liver cancer patients, and giving them a better way of life. However, further research on other animal models is warranted before their possible use in human beings. The efficacy as evidenced by so many parameters of studies also support the contention that the highly diluted form of potentized/dynamized remedies can actually act at the molecular level, as persistently being hypothesized by a group of Indian researchers in recent years by citing supportive controlled experimental evidences. The more research activities with an open mind will be carried out on this controversial science the more it will be enriched with the evidences to unravel its true flavour. An organised scientific search based on repeatable results obtained from authentic unbiased use of methodologies from basic sciences can say the final words. But till then, a relentless effort should be made to understand homeopathy converging both the philosophical and scientific rationalities. Hopefully, the works of the present thesis will stimulate further research in this direction.