A Dissertation Submitted to
THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY CHENNAI-600 032
In partial fulfillment of the requirements for the award of the Degree of MASTER OF PHARMACY
IN PHARMACOLOGY
Submitted By AARTHI.R
REGISTRATION No.261725901 Under the Guidance of Dr.C. JAIKANTH, M.Pharm., Ph.D.,
Department of Pharmacology
PSG COLLEGE OF PHARMACY PEELAMEDU
COIMBATORE 641 004 MAY-2019
Certificates
Dr. M. Ramanathan, M. Pharm., PhD., Principal & Head of the Department PSG College of Pharmacy, Peelamedu, Coimbatore - 641 004 (T.N)
`
CERTIFICATE
This is to certify that the dissertation work entitled “Investigation of growth inhibitory augmentation prospects of Brusatol with Paclitaxel in TNBC cell line” submitted by University Reg. No. 261725901 is a bonafide work carried out by the candidate under the guidance of Dr.C.JAIKANTH., M.Pharm., Ph.D., Associate professor, Department of Pharmacology, PSG College of Pharmacy and submitted to the Tamil Nadu Dr. M.G.R.
Medical University, Chennai, in partial fulfillment for the Degree of Master of Pharmacy in Pharmacology at the Department of Pharmacology, PSG College of Pharmacy, Coimbatore, during the academic year 2018-2019.
Dr. M. Ramanathan, M. Pharm., Ph.D., Principal & Head of the Department
PSG College of Pharmacy, Peelamedu, Coimbatore - 641 004
CERTIFICATE
This is to certify that the dissertation work entitled “Investigation of growth inhibitory augmentation prospects of Brusatol with Paclitaxel in TNBC cell line” submitted by University Reg. No. 261725901 is a bonafide work carried out by the candidate under my guidance and submitted to the Tamil Nadu Dr. M.G.R. Medical University, Chennai, in partial fulfillment for the Degree of Master of Pharmacy in Pharmacology at the Department of Pharmacology, PSG College of Pharmacy, Coimbatore, during the academic year 2018- 2019.
Dr C.JAIKANTH M.Pharm.,PhD., Associate Professor, Dept of Pharmacology,
I do hereby declare that the dissertation work entitled “Investigation of growth inhibitory augmentation prospects of Brusatol with Paclitaxel in TNBC cell line” submitted to the Tamil Nadu Dr.M.G.R. Medical University, Chennai, in partial fulfillment for the Degree of Master of Pharmacy in Pharmacology, was done by me under the guidance of Dr.C.JAIKANTH., M.Pharm., PhD., Associate Professor, Department of Pharmacology, PSG College of Pharmacy, Coimbatore, during the academic year 2018-2019.
University Reg. No; 261725901
This is to certify that the dissertation work entitled “Investigation of growth inhibitory augmentation prospects of Brusatol with Paclitaxel in TNBC cell line” submitted by University Reg. No. 261725901 to the Tamil Nadu Dr. M.G.R. Medical University, Chennai in partial fulfillment for the Degree of Master of Pharmacy in Pharmacology is a bonafide work carried out by the candidate at the Department of Pharmacology, PSG College of Pharmacy, Coimbatore and was evaluated by us during the year 2018-2019.
Examination Center: PSG College of Pharmacy, Coimbatore.
Date :
Internal Examiner External Examiner
To
My beloved Parents, Teachers
&
Almighty
CHAPTER NO
CONTENTS PAGE
NO
1 Introduction 1
2 Literature Review 5
3 Aim and Objective 37
4 Plan of Study 38
5 Materials and Methods 39
6 Results 47
7 Discussion 54
8 Conclusion 57
9 References 59
10 Annexure -
FIGURE
NO TITLE
PAGE NO
1 Normal breast tissue 5
2 Cellular process associated withanti cancer activity of quassinoids 27
3 Chemical structure of Brusatol 30
4 Hallmarks of cancer 34
5 Cell viability of BRU and PAC in MDA- MB- 231 cell line 47 6 Effect of BRU and PAC or their combination on cell viability 47 7 Apoptotic effect of BRU and PAC using AO-EB staining 49 8 BRU and PAC at concentration to promote inhibitory migration
effect on MDA-MB-231 cell 50
9 Caspase 3 activity determined by BRU and its combination 51
TABLE NO
TITTLE PAGE NO
1 Characterization of Breast cancer subtypes 8
2 Classification of breast cancer 15
3 Incidence of breast cancer worldwide 18
4 Incidence of TNBC in India 18
5 List of semi quassinoids anologue 28
6 Chemicals used 39
7 Primers used 39
8 Master mix composition 45
9 Master mix for PCR
46
Acknowledgement
Department of pharmacology, PSG college of pharmacy
ACKNOWLEDGEMENT
First and foremost I express bow down before lord Almighty for his blessings in completing my project work and throughout my life till this very second.
I owe my sincere thanks to my parents who cared for my well being and had spent their times in shaping my character, conduct and my life.
I would like to render my gratitude and special thanks to my beloved guide Dr.C. Jaikanth, M.pharm, Ph.D., Associate Professor, Department of Pharmacology, PSG College of Pharmacy, for this continuous support and his lavishing encouragement during my project.
It is my immense pleasure to be indebted to my beloved principal Dr. M. Ramanathan, M.
Pharm., Ph.D., for providing me the indispensable facilities and ideas to carry out the work successfully.
It is my pleasure to thank, Abdul Kayum, Research Scholar, Dept of Pharmacology for his selfless support during my project.
I would love to thank Mr.Mrinmoy Gautam, Research Scholar, Dept. of Pharmacology, for assisting me during my project work.
Besides my guide, I would like to thank Dr. Divakar, M.pharm, Ph.D.,and Mr.Ravikumar Rajan, Research Scholor, Dept. of Pharmacology, PSGCOP for their encouragement, and constructive ideas for the success of my project work.
It’s my pleasure to thank all other Staff members, Friends, Lab Technicians, library Persons and Lab Attenders for their help and support during my project work.
I would like to express my gratitude to the PSG Human ethics committee for their cooperation during my project work. I take this opportunity to thank ' PSG Sons’ and Charities for all the facilities that were provided to me at the institute enabling me to do the work of this magnitude.
I would also like to extend my special thanks to every single being who rendered their helping hands during my project work and also for being my stepping stones to the success of my project.
Introduction
Department of pharmacology, PSG college of pharmacy Page 1 1.INTRODUCTION
Breast cancer is a complex disease entity with different biological characteristics and clinical behaviour. According to American Cancer society “Breast cancer starts when cells in the breast begin to grow out of control. These cells usually form a tumor that can often be seen on an x-ray or felt as a lump. The tumor is malignant (cancer) if the cells can grow into (invade) surrounding tissues or spread (metastasize) to distant areas of the body. Breast cancer occurs almost entirely in women, but men can get breast cancer, too”.
Breast cancer is one of the most common cancers in the world and the foremost cause of cancer related death among women. As per GLOBOCAN 2012, 1.7 million new cases were diagnosed with breast cancer globally. While comparing with estimates of the year 2008, the incidence of breast cancer has increased by more than 20% and mortality rate has increased by approximately 14%. Moreover, it has evolved as an alarming cause of death due to cancer in less developed countries. In India, approximately 100,000 women are diagnosed with breast cancer with a case fatality ratio of 40%. This shows that India has become a country of high breast cancer related deaths worldwide(5)(Ferlay J et al.,2012).
Many clinical and pathological features have been defined to predict outcome and treatment response in breast cancer. These features include: Patient age, tumour stage, axillary lymphnode involvement, lymphovascular invasion, histologic grade, hormonal and human epidermal growth factor receptor (HER receptor) status. Breast cancer has been found to occur one in four of all the cancers in women. This is probably due to drastic changes in lifestyle of people and relatively poor clinical facilities to combat this disease(2)(Anderson KN et al.,2014).
The high occurrence of breast cancer among urban women can be correlated to several factors including having sex later, have fewer children, and breastfeed them less than the rural women which ultimately increases their exposure to oestrogen and subsequently risk of having breast cancer elevates greatly . Indian women also tend to have western diet which leads to obesity and high alcohol ingestion. Both of these factors contribute immensely in enhancing the risk of breast cancer(2)(Anderson KN et al.,2014).
Department of pharmacology, PSG college of pharmacy Page 2 The subtypes are characterized on the basis of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). These subtypes are as follows,
Luminal A (ER+/PR+HER2−, low Ki67 (<14%))
Luminal B (ER+/PR+, HER2+or HER2negative with high Ki67(>14%))
HER2 (ER−, PR−, HER2+)
Basal-like (ER−, PR−, HER2−)
Among the above classification triple negative breast cancer(TNBC)(ie basal like) is an aggressive subtype that is defined by lack of expression of ER and PR as well as absence of overexpressed or amplified HER2 (4)(Tang Y et al.,2016).
Triple negative breast cancer (TNBC) is characterized by an absence of the estrogen (ER) and progesterone (PR) receptors, as well as the human epidermal growth factor receptor 2 (HER2/HER) and accounts for about 20% of all breast cancer cases. The absence of the three receptors significantly reduces targeted treatment options for patients with TNBC and studies have shown that TNBC and HER-2 over-expression breast cancer have higher local recurrence and distant metastasis rates than other types (7)(Thakur KK et al.,2017).
Prevalence of TNBC in India ranged from 27% to 35% across studies, with a summary estimate of 31%. This is comparable to the prevalence seen in African American women and is more than twice the rate seen in white women. Some study also found a higher prevalence of premenopausal breast cancer, grade 3 disease, and larger tumor size, all of which are associated with triple-negative disease. Because TNBC is known to be more aggressive than other breast cancer subtypes, higher prevalence of TNBC could be a contributing factor to the high fatality rate of patients with breast cancer in India(6)(Sandhu GS et al.,).
Unlike hormonal receptor positive (HR+) and HER2 overexpressing breast cancers, TNBC is unresponsive to endocrine therapy and HER2-targeted agents and treatment options are limited to conventional cytotoxic chemotherapy. Chemotherapy has been effective in the treatment of early-stage disease, with pathologic complete response (pCR) rates exceeding those of HR+
subtypes. Patients with metastatic disease however experience rapid progression through several lines of chemotherapy, and overall survival (OS) in the metastatic setting is usually poor with
Department of pharmacology, PSG college of pharmacy Page 3 reports being between 9 and 13 months. Pathologic complete response rates to neoadjuvant chemotherapy (NACT) among patients with TNBC range from 27-45%, while pCR rate for patients with HER2 negative/HR+ breast cancer is generally around 10-20%. Pathological complete response has been proposed as a surrogate endpoint for prediction of long-term clinical benefit, such as disease free survival (DFS) and OS. However, while patients with TNBC who achieve a pCR appear to have a good DFS, patients with TNBC who have more than minimal residual disease at surgery have a much higher risk of early distant disease recurrence(27)(
Kumar P, Aggarwal R.2016).
Some of the treatment underwent for TNBC
Surgery
Radiotherapy
Chemotherapy
Limitations in TNBC{Citation}
Hormone receptor-positive tumors have been considered to have favorable outcome because of their response to endocrine manipulations such as tamoxifen, aromatase inhibitors, or ovarian ablation. The armory of “targeted therapy” for the treatment of metastatic TNBC has been inadequate due to the lack of identification of pathway specific targets and the absence of a validated targeted therapy. Patients with triple-negative breast cancer do benefit from chemotherapy, but better treatment options are needed that are less toxic, reduce the risk of disease progression, and are more targeted to this patient population (28)(Yao H et al.,2017).
Based on the fact that currently there are no approved targeted therapies for the neoadjuvant or palliative treatment of TNBC, identifying potential targets and developing effective targeted agents is greatly needed.
Also some studies has proved that the acquisition of metastatic potential during breast carcinogenesis and during chemotherapy is in part due to the epithelial-to-mesenchymal transition (EMT). During EMT, cancer cells go through the morphologic and phenotypic changes; increase in motility, dissemination, and dedifferentiation according to the signaling pathways that reprogram the gene expressions. The majority of signaling pathways and various
Department of pharmacology, PSG college of pharmacy Page 4 transcription factors, such as the Snail family, Twist, Zeb respond to these signals and function as master controllers of the EMT program (49)(Park SY et al .,2015).
In recent years, herbal medicines or natural compounds, either used alone or combined with conventional chemotherapeutic agents, have been shown to have beneficial effects on diverse cancers. Recent findings show that quassinoids are not just protein synthesis inhibitors, and that their anti-tumorigenic activity is likely associated with other properties. It has also been reported that quassinoids regulate DNA and RNA synthesis (45)(Uzoigwe J et al.,2012).
Brusatol (BR), a natural quassinoid diterpenoid isolated from Bruceae Fructus, exhibited the most potent in vitro tumor action among all the isolated quassinoids . Furthermore, it was reported that brusatol acted as a unique inhibitor of the Nrf2 pathway that sensitized various cancer cells and A549 xenografts to chemotherapeutic drugs, suggesting Brusatol might be a promising candidate for combating chemo-resistance and has the potential to be developed into an adjuvant chemotherapeutic agent (48)(Ren D et al.,2011).
Also studies has proved that Brusatol targeted inhibition of the EMT process without eliciting systemic toxicity using combination chemotherapeutic agents has lead to better tumor cell killing in Pancreatic cell line (51)(Lu Z et al.,2017).
On the other hand, paclitaxel being a potent chemotherapeutic agent for cancer , some studies have reported that paclitaxel-resistant TNBC cells have mesenchymal characteristics such as higher expression of mesenchymal markers like vimentin and combination therapy with paclitaxel and TGF-β receptor kinase inhibitors has proved to be a potential therapeutic strategy for TNBC(49)(Park SY et al .,2015).
Thus in our study, we hypothesize that Brusatol alone or in combination with chemotherapeutic agents such as Paclitaxel could suppress the expression of vimentin, while increase the expression of E-cadherin thus inhibiting the EMT process, and ultimately leading to the chemosensitizing effect of Brusatol.
In this study, we demonstrate a synergistic anti-tumor effect by combining Brusatol with Paclitaxel and determined that this occurred through their opposing effects on the EMT-MET axis in vitro.
Literature Review
Department of pharmacology, PSG college of pharmacy Page 5
2. LITERATURE REVIEW
2.1 BREAST CANCER
Breast cancer is a complex disease that displays a large degree of inter- and intratumoral heterogeneity. Breast cancer is a complex and heterogeneous disease and one of the leading causes of cancer-related death among women. According to American Cancer society
“Breast cancer starts when cells in the breast begin to grow out of control. These cells usually form a tumor that can often be seen on an x-ray or felt as a lump. The tumor is malignant (cancer) if the cells can grow into (invade) surrounding tissues or spread (metastasize) to distant areas of the body. Breast cancer occurs almost entirely in women, but men can get breast cancer too”. The breast is made up of lobes and ducts. Each lobe has many smaller sections called lobules. Lobules end in dozens of tiny bulbs that can make milk. The lobes, lobules, and bulbs are linked by thin tubes called ducts. When the cancer begins at cells of duct, it is known as ductal carcinoma and cancer that begins at lobes or lobules are known as lobular carcinoma.
These are said to be common types of breast cancer(1)( Yeo SK, Guan JL).
Fig1 Normal breast tissue
SOURCE: https://www.cancer.org/cancer/breast-cancer/about/what-is-breast-cancer.html
Department of pharmacology, PSG college of pharmacy Page 6 The uncommon type of breast cancer is inflammatory breast cancer in which the breast is warm, red, and swollen.
A tailored approach is necessary to elicit the best responses in patients when administering treatment modalities. Histological stratification of breast cancers based primarily on the expression of estrogen receptor (ER), progesterone receptor (PR), and ERBB2 receptor (HER2) has been useful and laid the foundation for the classification of breast cancers. In fact, breast cancer is a prototypic tumor in which the initial molecular subclassification has led to improved outcomes by guiding the administration of targeted therapeutics such as hormonal therapy (e.g., tamoxifen) and HER2-targeted therapy (e.g., trastuzumab). While histological stratification is still a common practice, technological advances have unraveled further complexities with the emergence of at least five distinct molecular subtypes [i.e., luminal A, luminal B, HER2- enriched, basal-like, and normal-like] based on gene expression clustering. Building on this, the combined genomic/ transcriptomic analyses of breast cancers have resulted in identification of 10 distinct breast cancer subtypes based on integrated clusters. In line with these advances, efforts to further segregate some of the established histological subtypes have also been carried out for ER negative and triple-negative breast cancers(4)( Tang Y et al.,2017).
The prognosis of breast cancer patients has been improved over time. However, further improvements in targeted treatment for breast cancer patients are expecting to solve the problem that why current therapy has effect only on a portion of the patients. A major milestone on the
TYPES OF BREAST CANCER
DUCTAL CACINOMA (common)
LOBULAR CARCINOMA
(common)
INFLAMMATORY
CANCER
(uncommon)
Department of pharmacology, PSG college of pharmacy Page 7 way to this goal is the definition of breast cancer molecular subtypes based on gene expression profiles: Basal-like, LuminalA, LuminalB, HER2-enriched and Normal-like (1) (Yeo SK, Guan JL).
2.2 BREAST TUMOR SUBTYPES
It is now widely recognized that breast cancer is not a single disease but one that is characterized by different subtypes determined by molecular and genetic information from tumor cells.
Prognostic differences in patient outcomes based on estrogen receptor (ER) and progesterone receptor (PR) status, as well as the expression level of human epidermal growth factor receptor 2 (HER2) have resulted in routine clinical stratification of tumors.(2) (Anderson KN et al 2014) Breast cancer is generally considered to be a highly heterogeneous cancer type, encompassing distinct phenotypic and morphological profiles, and thus possess very different clinical behaviors. Clinically, breast cancers are characterized into three basic types based on their immunohistochemical (IHC) properties (hormone status). They are hormone receptor-positive, HER2 positive (HER2+), and triple negative breast cancers. Hormone receptor-positive are the breast cancers with estrogen receptor-positive (ER+)/progesterone receptor-positive (PR+).
Approximately 85% of all breast cancers are hormone receptor-positive. Hormone receptor positive breast cancers can be further divided into two subtypes: Luminal A and Luminal B.
Luminal A tumors tend to be ER+ and/or PR+, and HER2-negative (HER2-). Luminal B tumors tend to be ER+ and/or PR+, and HER2+ (or HER2- with high Ki67). HER2+ refers to breast cancer that is human epidermal growth factor receptor 2 positive but hormone receptor-negative.
(4) (Tang Y et al.,2016).
Department of pharmacology, PSG college of pharmacy Page 8 Table1: Characterization of four major breast tumor subtypes, population prevalence, and clinical characteristics.
2.3 EPIDEMIOLOGY OF BREAST CANCER
With one million new cases of cancers reported in the World, breast cancer is common in females and comprises 18% of all women cancer. Incidence of breast cancer is predicted to increase to 85 per 100,000 women by 2021. In 2012, 1.67 million new cases of breast cancer were diagnosed, that is 25% of all cancers among women. Studies stated that 883,000 cases are in less developed countries and 794,000 in most developed countries. According to the data, 145.2 women in Belgium and 66.3 in Poland between 100,000 suffer from breast cancer.
Incidence of breast cancer in the United States is one out of eight women and in Asia one woman suffers from breast cancer out of 35. In Iran, there are 10 cases in 100,000 populations and 7000 new cases have been reported annually. Prevalence of breast cancer is increasing in Pakistan.
Breast cancer is found mostly in highly populated areas of South Asian developing counties.
Breast cancers in males have been detected in Northern areas of Pakistan. Yang et al stated that new cases of breast cancer in China were 168,013 in 2005 and 121,269 in 2000.
Incidence of breast cancer has been increasing worldwide, but remarkably distinct differences have been observed between rich and poor countries. In Western Europe, breast cancer incidence has reached ≥90/100,000 whereas it is 30/100,000 women annually in eastern-Africa. However,
Department of pharmacology, PSG college of pharmacy Page 9 breast cancer mortality rates in these two regions are almost the same which is about 15/100,000, clearly denoting late diagnosis and poor survival in eastern-Africa. (5)( Ferlay J et al.,2012).
2.4 CAUSATIVE FACTORS AND ASSOCIATED RISKS OF BREAST CARCINOMA Breast cancer is more common in single women than in married women. The breast is an estrogen sensitive organ. Many females who have been on birth control pills or estrogen replacement have found that the medications result in enlarged and often tender breasts. The activity of this medication, combined with the standard western high fat, low fiber diet, which over-stimulates breast tissue, could be a trigger for breast cancer. Epidemiological investigations have also suggested that those women who have many children possess lower risk of breast cancer than those women who have fewer children. Incidence of breast cancer is 10.04% among all cancers and, most commonly occurs in 40–50 aged women(11)( Shamsi U et al.,2014).
Depression is most commonly found in women with breast cancer(12)( Donnelly T et al.,2014).
Andsoy et al. conducted a study to investigate knowledge of colorectal, cervical and breast cancer. (14)( Franca A et al., 2012).
Menopause resulting from surgical removal of ovaries (oophorectomy) decreases the risk.
Presence of certain kinds of benign tumours in breast increases the risk of malignancy. The ovaries stop producing the female hormones once the menopause sets in, but in obese women the fatty tissue can provide the estrogen as it is capable of producing it. This increase in hormone production seems to increase the risk of breast cancer in obese post menopausal women(15)(
Kruk J et al.,2007).
Deficiency of vitamin D and lack of sun exposure is considered to be the important cause of breast cancer. It is found to be more in women than men. The risk of breast cancer increases with age however rarely found before the age of 20 years. Carcinoma in one breast can increase the risk by four times in another breast(16)(Alco G et al., 2014). While the patients that have the history of ovarian, endometrial or colon cancer have 1–2 times increased risk to develop breast carcinoma. A female who has had breast cancer has an enhanced danger of occurring breast cancer in the other breast(17)( Kelsey J et al., 1996).
Department of pharmacology, PSG college of pharmacy Page 10 The minimal role of the gene has been established in the development of breast cancer. BRCA-1 (breast cancer susceptibility gene) is considered to be the cause of 5–10% of breast cancer that is transferred from either father or mother to the next generation. The study indicates that right environmental conditions are required for cancer promoting gene for expression. Certain families have been recognized with a genetically higher incidence of early onset breast cancer. Women who have a positive family history of breast carcinoma are 2–4 times more likely to develop the cancer, especially the females who are the carriers of BRCA1 or BRCA2 genes have the significant chance to develop carcinoma of breast(18)( McPherson K et al., 2000).
Steroid hormones include androgens, progesterone and estrogen, which belong to a cluster of structurally connected hormones known as sex hormones that are released into the blood by the gonads and adrenal glands. Oestrogens have important activities on differentiation, growth and performance of several tissues, including urogenital system of man and women, cardiovascular system, brain, uterus and breast. Fat cells are found in excess amount in breast of aged females;
therefore, the quantity of estradiol is higher in breast tissues of post menopausal female than their plasma level. This most likely is responsible for the increasing occurrence of breast cancer in aged female and assists the action of steroid hormones in breast cancer pathogenesis.
Benign tumors and proliferative lesions without or with atypia can increase the risk of breast cancer (19) (Weinberg O et al.,2005). Breast cancer has been linked with high level of dietary fats and low level of certain nutrients for various years. Animal fat stimulates colonal bacterial to form estrogen from cholesterol found in the diet, thus increasing level of estrogen in the body.
The body fat is also involved in synthesis of oestrone, a type of estrogen.
2.5 STAGES OF BREAST CANCER
According to the report of breast cancer.org Stages of the breast cancer depends upon the size and type of tumor and how much the tumor cells have been penetrated in the breast tissues.
Whereas stage 0 describes the non invasive and stage 4 describes the invasive kind of tumor.
Department of pharmacology, PSG college of pharmacy Page 11 Descriptions of those tumor stages are:
Stage 0
This is the non invasive stage of tumour which indicates that both cancerous and non cancerous cells are within the boundaries of that part of the breast in which the tumor begins to grow and no evidence found of their invasion in the surrounding tissues of that part, the example of this tumour stage is ductal cell carcinoma in situ (DCIS).
Stage 1
This stage describes as the invasive breast carcinoma and microscopic invasion is possible in this stage. It has two categories that are 1A and 1B stage. The category 1A describes the tumor which measures up to 2 cm and none of the lymph nodes are involved in it while stage 1B describes that small group of cancer cells larger than 0.2 mm founds in lymph node.
Stage 2
Stage 2 also has two categories 2A and 2B. Stage 2A describes that the tumour is found in axillary lymph nodes or in sentinel lymph nodes but no tumor found in breast. The tumor can be smaller or larger than 2 cm but not more than 5 cm. However stage 2B describes that the tumor could be larger than 5 cm but can’t reach to the axillary lymph nodes.
Stage 3
It has been divided into three sub categories that are 3A, 3B and 3C. Amongst which stage 3A describes that no tumor is found in breast but it can be found in 4–9 axillary lymph nodes or in sentinel lymph nodes while stage 3B describes that the tumour can be of any size but have caused swelling or ulcer on the skin of the breast and can have spread up to 9 axillary lymph nodes or to sentinel lymph nodes stage 3B can be considered as inflammatory breast cancer which includes red, warm and swollen skin of the breast. However stage 3C describes the spread of tumor up to 10 or more than 10 axillary lymph nodes and it also have involved the lymph nodes above and below the clavicle.
Department of pharmacology, PSG college of pharmacy Page 12 Stage 4
This is the advanced and metastatic stage of cancer and this stage describes the spread to other organs of the body that is lungs, bones, liver brain etc.(3)
2.6 TREATMENT
Breast cancer management strategies differ depending on the step of the cancer—its mass, place, whether it has extended to other organs of the body and the physical condition of the individual.
Present management for breast cancer includes targeted therapies, hormonal treatment, radiation therapy and surgery.
Surgery
This is the foremost management strategy for individuals whose breast cancer has not extended to further areas of the body and is also a choice for further complex stages of the illness. The kinds of breast cancer surgery vary in the quantity of tissue that is excised with the cancer; this depends on the cancer’s characteristics, whether it has extended, and the patient’s special feelings. A few of the most familiar kinds of surgery include:
Lumpectomy
Mastectomy
Reconstructive surgery
Role of estrogen and progesterone receptors in the management of breast cancer
The estrogen receptor assay has developed into a typical practice in the treatment of complex breast cancer. Tumors missing estrogen receptor react occasionally to endocrine treatment, while improvement proportions of 50–60% are seen in estrogen receptor positive tumors. Current researches demonstrate that the estrogen receptor condition of the principal cancer is a superior interpreter of the endocrine reliance of metastatic cancers at the moment of clinical deterioration.
Department of pharmacology, PSG college of pharmacy Page 13 Anti‑estrogen therapy
It can be used in such types of cancers that are affected by hormones and the tumor has hormone receptors such as estrogen receptors. The most common category of drugs that are used in breast cancer is anti estrogen, which includes the agents that are (tamoxifen, raloxifene, toremifene etc).
Tamoxifen inhibits the hormone oestrogen from entering into cells of the breast cancer. This mechanism inhibits the breast cancer cells from developing. Tamoxifen can be suggested to treat female of any age group. However tamoxifen is considered as the drug of choice in women that have positive estrogen receptor breast carcinoma. Tamoxifen is a selective estrogen receptor modulator (SERMS) and acts like estrogen on other parts of the body such as uterus. However, it demonstrates anti estrogen properties of breast tissues and competes with estrogen for binding to the estrogen receptors in the breast.
Radiation therapy
It is useful for reducing the necessity of mastectomies. A combination of a lumpectomy and radiation therapy is being increasingly used over a mastectomy in the early stages of breast cancer. Use of radiation therapy may be done after breast cancer surgery to destroy the remaining cells in the chest area.
Chemotherapy
The process of killing cancer cells by using certain medicines is termed as chemotherapy. It can be given in both situations, before and after surgery, depending upon the condition of the patient.
According to the American cancer society the medicines include in chemotherapy are Docetaxel, Paclitaxel, Platinum agents (cisplatin, carboplatin), Vinorelbine (Navelbine), Capecitabine (Xeloda), Liposomal doxorubicin (Doxil), Cyclophosphamide (Cytoxan), Carboplatin (Paraplatin) etc. However it has various side effects. Metastatic or secondary breast can is difficult to treat but it can be controlled and sometime for various years. Chemotherapy can be prescribed to manage metastatic breast cancer to minimize or sluggish its development. It can also be administered to decrease some manifestations. Other treatment option can be initiated prior or alongside chemotherapy.
Department of pharmacology, PSG college of pharmacy Page 14 Anthracyclines
Anthracycline are commonly prescribed in the treatment of breast cancer. They impede with enzymes associated the DNA copying, which is desired for cells to separate to create new cells.
Epirubicin and doxorubicin are the most commonly used medicines in breast cancers. There is proof that anthracyclines functions better than various other chemotherapy medicines.
Immunotherapy
It utilizes the immune system of the body to fight against the cancer cells. Cancer vaccine is one of its examples. Parts of cancer cells or cancer cells are utilized for formation of vaccines. These cells excite the body’s immune system to assist assault and destroy cancer cells . Immunotherapy has turn into a significant constituent in the management of breast cancer. HER2 targeted treatment are at the present an important part of HER2 over expressing breast tumor therapy.
Trastuzumab, with the new current accompaniments of pertuzumab and TDM1, encompass considerably superior breast cancer prediction. With various Federal Drug and Administration recommended antibody treatments used in together the adjuvant and metastatic settings, development progresses to be done in the area of immunotherapies. (3) (Akram M et al., 2017) Type IHC
characteristics
Clinical features Treatment Resistance
Luminal A
ER+/PR+, HER2−, low Ki67 (<14%)
~40% of invasive breast cancers;
best prognosis among all four types
endocrine therapy;
chemotherapy (less
responsive than in
luminal B type);
tamoxifen resistance;
multidrug resistance (MDR);
de novo or acquired,
mostly acquired
Luminal B
ER+/PR+, HER2+, (or
~20% of invasive breast
endocrine therapy
tamoxifen resistance;
Department of pharmacology, PSG college of pharmacy Page 15 HER2
negative with high Ki67) (>14%)
cancers; Higher grade
than luminal A;
good prognosis although not as good as Luminal A
(less responsive than in Luminal A
type);
chemotherapy
multidrug resistance (MDR);
de novo or acquired,
mostly acquired
HER2 ER−, PR−,
HER2+
15% ~ 20% of invasive
breast cancers;
high
grade; lymph node
positive; better prognosis
than triple negative but worse than luminal type
HER2 targeted therapy;
anthracycline- based
chemotherapy;
trastuzumab resistance;
multidrug resistance (MDR); de novo or acquired, mostly acquired
Basal-like
ER−, PR−, HER2−
10% ~15% of invasive
breast cancers;
BRCA1 dysfunction;
worst prognosis among
all four types
platinum-based chemotherapy and
PARP inhibitors;
multidrug resistance
(MDR); de novo or
acquired, mostly acquired
Table2: classification of breast cancer
Department of pharmacology, PSG college of pharmacy Page 16 2.7 Triple Negative Breast Cancer
Triple-negative breast cancer (TNBC) is a disease characterized by the lack of estrogen receptor (ER) and progesterone receptor (PgR) expression as well as human epidermal growth factor receptor 2 (HER2) amplification, and accounts for 10–20% of all breast cancers. Recent progress in targeted therapies, both in adjuvant and metastatic settings, has improved the prognosis of ER positive and HER2-positive breast cancers, whereas the improvement in survival has currently plateaued for TNBC. Although conventional cytotoxic chemotherapy based on anthracyclines and taxanes is effective for a subset of patients with TNBC, some cases show a very aggressive clinical course. TNBC is associated with a higher rate of distant recurrence and a poorer prognosis than those in patients with other breast cancer subtypes, and fewer than 30% of patients with metastatic TNBC survive five years Hence, there is an immediate need to develop novel therapeutic strategies for TNBC(52)( Oba T, Ito KI et al., 2018).
Triple negative is a phenotype and its major components in molecular assays are the basal-like tumors, normal breastlike tumors and the more recently recognized, the uncommon but intriguing, claudin-low molecular subtypes. It also includes BRCA1-deficient breast tumors. Basallike breast cancers were so named because the neoplastic cells of this tumor type consistently express genes usually found in normal basal/myoepithelial cells of the breast. These account for 15 % of all breast cancers and have several molecular characteristics including low signals for the ER-related gene cluster and HER2-related gene clusters.
2.8 INCIDENCE OF TNBC
Gradually, the burden of TNBC among women is increasing globally and this becomes an issue of prime attention. The table summarizes the data on TNBC patients obtained from numerous clinicopathological studies published till date and categorizes them based on region, age, number of breast cancer patients reported and percentage of TNBC patients. (6)(Sandhu GS et al.,2016).
Department of pharmacology, PSG college of pharmacy Page 17 COUNTRY/CITY PERIOD OF
DIAGNOSIS
AGE NO. OF
PATIENTS
TNBC PERCENTAGE REFERENCE
Algeria
(North Africa)
2008-2013 Mean age ( 46.9 YRS)
3014 627 20.80% Chebral.et
al.2015
Spain (Europe)
1998-2012 Median age 32 years
341 57 17% (Zugazagoitia
et al, 2014)
Germany (Europe)
2008-2011 Mean age 55.9 years (range:
29-93 years)
315 33 10.5% (Wojcinski et
al, 2012
Boston (USA)
1998-2006 415
Median age 58 years
415 81 19.5% (Stead et al,
2009)
Karachi, Pakistan (South Asia)
2010-2012 Mean age 48.4 years
1104 205 18.6% (Hashmi et al, 2014)
Department of pharmacology, PSG college of pharmacy Page 18 Table :3 Incidence of breast cancer worldwide
2.9 INCIDENCE OF TNBC IN INDIA
In India, approximately 100,000 women are diagnosed with breast cancer with a case fatality ratio of 40%. This shows that India has become a country of high breast cancer related deaths worldwide. Currently, TNBC is highly prevalent among Indian women and occurs in about 20- 43% of all patients with breast cancer. Table shows that the incidence of TNBC is highest (27.9%) in India as compared to other regions of the world. (6)(Sandhu GS et al.,2016).
city/ area Period of doagnosis
Age No. Of
patients
TNBC Percentage
%
Ref
Srinagar (North India)
2009-2013 Median age 47years (range: 23-70 years)
180 62 34.4% (Nabi et al, 2015)
New Delhi 2008-2011 Median age 50 years
1284 306 23.8% (Doval et al, 2015)
Bengaluru (South India)
2011-2014 Median age 49 years (range: 24-75 years)
100 20 20% (Geethamala
K, et al, 2015)
Netherland (Europe) India (South Asia) 2004-2013 Mean age 49.8
years
7002 1951 27.9%
1989-2008 8752 58 6 6.7% (Kwast et al,
2012)
Department of pharmacology, PSG college of pharmacy Page 19 Mumbai
(Western India)
2009-2010 Median age 50 Years (range: 19-88 years)
206 82 39.8% (Singh et al, 2014b)
Hyderabad (South India)
2001-2007 Mean age 50 years (range:
20-87 years)
605 138 22.8% (Zubeda et
al, 2013)
Chennai South India)
2009-2010 median age 53.8 years
(range: 24-99
321 81 25.2 % (Ambroise
et al, 2013)
REF: PMID: 28801156
Table4: incidence of TNBC in India
The main characteristics of triple-negative breast cancers are as follows:
(a) frequently seen in younger women (\50 years)
(b) are more frequent in African-American women and black ethnicities (c) usually present as interval cancers
(d) have high chemosensitivity,
(e) have a weak association between tumor size and lymph node metastases
(f) are more aggressive with higher chance of brain metastases and high chance of recurrence during the first and third year after diagnosis
(g) have shorter survival following first metastatic event when compared with other subtypes.
(28)
Department of pharmacology, PSG college of pharmacy Page 20 2.10 ASSESSMENT OF ER, PR AND HER2 STATUS
IHC is currently the predominant method for determination of ER, PR and HER2 status, often with additional FISH assays to clarify HER2 immunohistochemical results, but differences in methods and interpretation can substantially affect the accuracy, resulting in misclassification.
Many factors influence the accuracy of detection of ER, PR and HER2 in the laboratory. These include preanalytical factors (including prompt fixation of appropriate duration), analytic factors (including utilization ofvalidated procedures with ongoing proficiency testing) and post-analytic factors (including appropriate reportingThe American Society of Clinical Oncology (ASCO)/College of American Pathologists guidelines for IHC testing for ERs and PRs recommend that ER and PR assays be considered positive if there are at least 1 % of positive tumor cells in the sample . HER2 receptor assessment still follows a standardized definition according to the guidelines of the joint committee of the ASCO/College of American Pathologists, which include the following:
(a) recommendations for tissue fixation for more than 6 and less than 48 h;
(b) new scoring criteria, including a new threshold of 30 % strong immunostaining for classification of 3+ ;
(c) introduction of the term ‘equivocal’ to characterize HER2 studies that are 2+ by IHC and/or show HER2/chromosome 17 ratios of between 1.8 and 2.2 by FISH;
(d) requirements for laboratories to validate HER2 assays, generally through the cross-testing of cases with another HER2 methodology, with laboratories required to attain 95 % concordance for both positive and negative tests; and
(e) participation in HER2 proficiency testing.
The adoption of a standardized definition for hormone receptor positivity worldwide would enable better definition of patients with TNBC and thus improve the quality of research conducted on this patient subset. In this respect, the need for an accurate and reproducible assessment of triple-negative status by pathologists cannot be overemphasized. (29)( Harvey JM et al.,).
Department of pharmacology, PSG college of pharmacy Page 21 2.11 EPIDEMIOLOGY AND RISK FACTORS
The risk of developing TNBC varies with age, race, genetics, waist hip ratio, breastfeeding patterns and parity. Bauer et al found that women with TNBC were significantly more likely to be under the age of 40 years. Regardless of the stage at diagnosis, these women had poorer survival than those with other breast cancers. Rauh et al. confirmed that age at diagnosis was an important factor influencing the distribution of molecular subtypes; young patients aged 42 years or less and perimenopausal patients with a body mass index (BMI) more than 32.4 kg/ m2 more often developed TNBC. In a retrospective study conducted by Vona-Davisetal found that TNBC appeared more frequently in younger women, with later stage at diagnosis and in association with obesity. Kwan et al. also confirmed that women with TNBC were more likely to be overweight or obese if premenopausal. Although TNBC is a hormone-insensitive disease, several hormonal alterations throughout a woman’s life are associated with an increased risk of developing TNBC. Multiparity and young age at first pregnancy increase the risk of developing TNBC, but breastfeeding, a longer duration of breastfeeding and an increasing number of children breastfed, all reduce the risk of developing TNBC. A higher waist/hip ratio is also associated with an increased risk for developing TNBC. Oral contraceptive usage for more than 1 year was also found to be associated with 2.5-fold increase risk of TNBC and no significantly increased risk of non-TNBC. Age at menarche and age at menopause were also found to be modestly associated with increased risk of ER-positive but not TNBC subtype. Kabat et al concluded that cigarette smoking was not associated with TNBC, whereas alcoholism decreased the risk of TNBC compared with non-alcoholics. It was also suggested that triple-negative or basallike tumors might have distinct etiology and accounted for much of the risk factor heterogeneity between hormone receptor-positive and hormone receptor-negative tumors. A recent study found that women with triple-negative tumors had a high prevalence of metabolic syndrome, characterized by obesity and insulin resistance. Insulin resistance may increase the risk of premenopausal breast cancer by reducing sex hormone-binding globulin levels, resulting in an increase in free estrogen and androgen levels, and increasing proliferation of breast epithelial cells(32)( Lakhani SR et al.,).
Department of pharmacology, PSG college of pharmacy Page 22 2.12 TNBC AND GENETICS
Little is known about the etiological factors at the genetic level which promote the initiation and development of TNBC. Patients with hereditary breast and ovarian cancer syndromes have a 50– 85 % risk of developing breast cancer during their lifetime. Germline mutations in the BRCA1 breast and ovarian cancer susceptibility gene have been associated with TNBC, with 60–80 % of breast tumors from BRCA1 mutation carriers displaying a TNBC phenotype. The studies concluded that deleterious mutations in predisposition genes were found at high frequency in patients with TNBC unselected for family history of cancer, and patients with TNBC, regardless of age at diagnosis or family history of cancer, should be considered for germline genetic testing of BRCA1 and BRCA2(34)( Comen EA et al.,2007).
2.13 PROGNOSTIC FACTORS AND PROGNOSIS
There is difference in outcomes within different phenotypes of breast cancer and the recurrence pattern of TNBC or basal-like cancers is different as compared to ER-positive tumors. For TNBC, relapse rates are high during the first few years following surgery with a peak recurrence risk at 3 years after surgery; thereafter, recurrence risk rapidly decreases. In contrast, more than 50 % ER-positive breast cancers recur between 5 and 10 years after surgery, and in the long term more events will occur in high-grade ER-positive breast cancers as compared to TNBC. The studies summarized the observations as:
(a) TNBC often presented as interval cancer,
(b) there is a weak relationship between tumor size and node status, (c) there was rapid rise in the risk of recurrence following diagnosis, (d) the peak risk of recurrence was at 1–3 years,
(e) distal recurrence was rarely preceded by local recurrence, (f) local recurrence was not predictive of distal recurrence, (g) there was increased mortality rate in the first 5 years, (h) the majority of deaths occurred in the first 5 years, and
Department of pharmacology, PSG college of pharmacy Page 23 (i) there was rapid progression from distant recurrence to death.
Some data suggest that both TNBC and HER2-positive breast cancers are at increased risk for locoregional relapse even after mastectomy. The risk of developing subsequent distant metastasis and death following locoregional recurrence is higher in TNBC compared to other subtypes.
TNBC is associated with higher expression of Ki67 and p53 compared with non-triple negative (TN) tumors, which may contribute to poor prognosis in TN tumors(38) (Perou CM, Sorlie T, Eisen MB et al 2000). It might therefore be reasonable to assume that the correlation of Ki67 with breast cancer outcome involves a mixture of prognostic and predictive effects. (40)( Jatoi I et al.,2011).
2.14 CHARACTERISTICS OF TNBC Gene expression profiling of TNBC
The heterogeneity of TNBCs in terms of gene expression profiling and responses to therapeutic regimens is widely acknowledged. A study by Lehmann et al. has revealed 6 distinct subtypes of TNBCs defined by their gene expression profiles. These subtypes identified are
basal-like 1 and 2 (BL1 and BL2), immunomodulatory (IM),
mesenchymal (M),
mesenchymal stem-like (MSL), and luminal androgen receptor (LAR)
They differ in important biological pathways and prognosis. For example, BL1 and BL2 were highly proliferative and had a higher expression of the genes related with cell-cycle and DNA damage response. The M and MSL groups were enriched for genes of the epithelial-to- mesenchymal transition (EMT) pathway, whereas the IM subtype was characterized by immune cell signaling features. The LAR subtype was ER-negative but AR-positive and the LAR cell lines were sensitive to the AR antagonist bicalutamide. This sub-classification of TNBC is useful not only in the understanding of the disease properties but also in the identification of the molecular targets for treatment.
Department of pharmacology, PSG college of pharmacy Page 24 2.15 THERAPEUTIC OPTIONS
Generally speaking, patients with TNBC are treated similarly as women who present with non- TNBC, especially in terms of adjuvant and neoadjuvant settings. Surgery and radiotherapy are employed routinely in a similar way as with other types of breast cancer. Patients with TNBC do not benefit from therapies that are designed to target the hormone receptors (such as tamoxifen) or HER2 (such as Herceptin). Currently, chemotherapy, individually or in combination with surgery and/or radiotherapy, is the standard treatment mode for TNBC. TNBCs can be chemo- sensitive particularly to cytotoxic agents, such as anthracyclines and taxanes, which are part of the standard therapy used for high-risk patients, for example patients with lymph node-positive disease(41) (Isakoff SJ 2010).
Cytotoxic chemotherapy
Currently, taxane- and anthracycline-based combination chemotherapy remains the standard treatment approach for early-stage TNBC patients, and this approach has changed little during the last decade. To date, there are no specific guidelines for chemotherapeutic management of TNBC. The European Society for Medical Oncology (ESMO) states that cytotoxic chemotherapy is the standard of care for the treatment of TNBC and that the choice of the regimen should be made after consideration of disease related factors (previous therapies and response, tumor burden, and need for rapid disease/symptom control) and patient-related factors (patient preferences, biological age, menopausal status, comorbidities and performance status, and socioeconomic and psychological factors). Microtubule stabilizers Microtubule stabilizers (such as taxanes) are a group of potent tubulin polymerizers that are available for the treatment of breast cancer. Many studies have demonstrated that taxanes (paclitaxel [Taxol], docetaxel [Taxotere], cabazitaxel) are more effective for TNBCs than receptor-positive cancers. Although the role of anthracyclines alone in TNBC is questionable, a definite benefit was observed when anthracyclines were used in combination with taxanes in node-positive TNBC patients. Platinum agents The intense interest in the role of platinum compounds including carboplatin and cisplatin in TNBC is based on phenotypic and molecular similarities between BRCA-associated breast cancer and the basal-like subtype.
Department of pharmacology, PSG college of pharmacy Page 25 Targeted therapies
PARP inhibitors Poly (ADP-ribose) polymerase (PARP) plays a vital role in the repair of single- stranded DNA breaks through the base excision repair pathway. As mentioned above, TNBC has a high frequency of mutation of the breast cancer susceptibility gene BRCA. Therefore, PARP inhibitors can lead to cell death in BRCA-mutated TNBCs because of the inability of the cell to repair DNA damage due to BRCA mutation. It has been demonstrated that PARP inhibition potentiates the effects of ionizing radiation agents, DNA-methylating compounds, topoisomerase inhibitors, and platinums. Several PARP inhibitors such as olaparib (AZD 2811) and BSI-201 are at different stages of clinical development.
Angiogenesis inhibitors
Expression of vascular endothelial growth factor (VEGF) is much higher in TNBC compared with nonTNBC. Bevacizumab (Avastin), an anti-VEGF monoclonal antibody, has consistently exhibited improved PFS and response rate when used in combination with first-line chemotherapy in HER2-negative breast cancer. (42)( Gronwald J, Byrski T, Huzarski T et al 2009).
Statins
Statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, reduce the intracellular biosynthesis of cholesterol by reversibly inhibiting the conversion of HMG-CoA to mevalonate. Recently, statins have also pleiotropic anti-cancer effects in a variety of cancers including breast cancer. Preclinical studies have shown anti-proliferative, pro-apoptotic, anti- invasive, and radio- and chemo-sensitizing properties of statins. Given that statins are FDA- approved, well tolerated, and affordable, they provide the opportunities for accelerated repurposing as cancer therapeutics. The molecular mechanisms underlying lovastatin’s effect on MDA-MB-231 cells included modulation of the proteins involved in apoptosis, differentiation, cell proliferation, signal transduction, epithelial-to-mesenchymal transition (EMT) and tumor metastasis. Therefore, statins have the potential to prevent or treat a subset of breast cancers, such as TNBC(43) (Byrski T, Huzarski T, Dent R et al 2009).
Department of pharmacology, PSG college of pharmacy Page 26 In spite of the general susceptibility to standard chemotherapy, TNBCs exhibit an overall poorer survival compared to non-TNBCs. The benefits of targeted therapies have eluded patients with TNBC due to the absence of well-defined molecular targets. Novel therapeutic targets that are being actively explored and new agents with therapeutic potential that are under development.
Two important areas need in-depth investigations that may bring about significant changes in the management of TNBC.
First, the identification of molecular targets will be crucial to identifying actionable targets in patients with TNBC.
Secondly, agents that selectively or preferentially target TNBC are urgently needed(27)( Kumar P, Aggarwal R 2017)..
2.16 QUASSINOIDS AND ANTI-CANCER ACTIVITIES
Natural products have been a popular source of medicinal agents for thousands of years. The quassinoids are a class of inhibitor for eukaryotic protein synthesis is well supported, although how the distinct compounds exert this effect is still not fully understood. The ability to interfere at the peptidyl-transferase site, thus preventing peptide bond formation and terminating chain elongation, has been proposed as the major mechanism responsible for antineoplastic activity.
Recent findings show that quassinoids are not just protein synthesis inhibitors, and that their anti- tumorigenic activity is likely associated with other properties. (44) (Fiaschetti G et al.,2011).
Quassinoids represent a heterogeneous group of molecules with the ability to interfere with different pathways implicated in tumorigenesis. Several apoptosis-related processes have been found altered upon treatment with members of this class of compounds. For instance, both bruceantin (1) and brucein-D (2) induce the proteolytic activation of caspase-3, -8 and -9 and proteolytic processing of BID. However, brucein-D induces cell death via a complex mechanism involving GSH depletion, oxidative stress and p38-MAPK activation, together with prevention of NF-B translocation into the nucleus and the consequent inhibition of antiapoptotic gene expression. It is interesting to note that the quassinoid, brusatol (3), induces an opposite effect, promoting NF-B translocation into the nucleous via induction of phosphorylation of IB. These diverse effects on the NF-B pathway may also reflect differences in the cellular models used to study the mechanism of action of the two quassinoids(44)(Fiaschetti G et al.,2011).
Department of pharmacology, PSG college of pharmacy Page 27 Fig 2: Cellular processes associated with anticancer activity of quassinoids
2.17 SEMI-SYNTHETIC QUASSINOID ANALOGS
Semi-synthesis plays a major role in the structural modification of natural products, and can lead to the discovery of more active and/or less toxic compounds. Semisynthetic procedures to modify quassinoid molecules are presently well established. The first investigations on semi- synthetic quassinoids involved dimeric derivatives. Connecting two molecules of bruceantin or brusatol at the C-3 hydroxy group through malonate, glutarate, adipate and sebacate esters, Lee et al. generated bis-esters active against leukemic cells (P-388). Brusatol dimers had higher potency as antineoplastic agents than brusatol itself, while brusatol was more active as an anti- inflammatory or differentiating agent.
Department of pharmacology, PSG college of pharmacy Page 28 Table 5: List of semi synthetic quassinoids anologue
2.18 IMPORTANCE OF COMBINATION CANCER THERAPY USING NATURAL COMPOUNDS
Optimally, cancer is prevented, and when it occurs, treated effectively with as little attendant toxicity as possible. The prevention and treatment of cancer through intervention with one or more natural compounds has received increasing attention. Combination therapy involves the simultaneous use of two or more agents. Monotherapy has limited efficacy in the treatment of most adult malignancies owing to the multiple target nature of the disease, and limited efficacy in prevention because of the heterogeneous pathways by which cancer can form . Combination chemotherapy has been practiced at least since the 1960s. Normal tissue toxicity often limits the use of a single agent. Natural compounds are more likely than pharmacologic agents to be associated with toxic effects other than dose, including adulteration, substitution, contamination and lack of standardization. Preparation of natural compounds for consumption requires the same
Department of pharmacology, PSG college of pharmacy Page 29 rigor in preparation as pharmacologic agents. Combination therapy to treat cancer has taken the form of multiple chemotherapeutic agents and more recently as chemotherapy plus targeted biologic therapy. There is increasing evidence that the isolation of a single compound from complex foods may not be effective in preventing cancer even when administered at toxic doses, whereas combination therapy using lower doses with no or lower toxicity might work. Targeting multiple pathways, or the same pathway through a different mechanism, maximizes the chance for effect while limiting normal tissue toxicity(45)( Uzoigwe J, Sauter 2012).
Combination therapy interactions
Drug interactions are exploited in combination therapy. These interactions can be either beneficial or problematic. The observed interactions can provide clues as to how the agents work: synergistic interactions suggest different sites of action, while additive interactions suggest a common site of action.
Synergistic
This occurs when the combined activity of two or more compounds produces a greater than simply additive effect. An advantage of drug synergy is that it allows a therapeutic effect to be achieved with low doses of component interventions, which often minimizes potential side effects.
Additive
If two interventions act additively, then the effect of the combination of half the dose of each agent produces the same effect as a full dose of one parent agent.
Antagonistic
Antagonistic interaction is observed when the combined activity of two or more compounds is less than expected given their individual activities. Plant & animal compounds to treat or prevent cancer Plant and animal compounds are associated with important health benefits, including anticancer properties. Indeed, many natural compounds have been used in combination, primarily in preclinical studies, in order to determine their efficacy in the treatment or prevention of cancer. Such compounds include plant-derived (e.g., polyphenols, phytosterols, triterponids
Department of pharmacology, PSG college of pharmacy Page 30 and saponins), animal-derived (e.g., vitamin D and omega-3 fatty acid) and plant- and animal- derived (carotenoid) compounds(45)( Uzoigwe J, Sauter.,2012).
2.19 BRUSATOL
Brusatol (BR; C26H32O11), the chemical structure of which is presented in, is one of the major quassinoids isolated from B. javanica. This compound has been reported to exert marked anti‑inflammatory, antimalarial and antitumor activities. In addition, BR has been demonstrated to uniquely block the nuclear factor erythroid 2-related factor 2 pathway, thus sensitizing various cancer cells in vitro and A549 mouse xenografts to chemotherapeutic agents,
Fig 3: Chemical structure of brusatol.
In recent years, herbal medicines or natural compounds, either used alone or combined with conventional chemotherapeutic agents, have been shown to have beneficial effects on diverse cancers. Brusatol (BR), a natural quassinoid diterpenoid isolated from Bruceae Fructus, exhibited the most potent in vitro antipancreatic tumor action among all the isolated quassinoids.
Furthermore, it was reported that brusatol acted as a unique inhibitor of the Nrf2 pathway that sensitized various cancer cells and A549 xenografts to chemotherapeutic drugs, suggesting brusatol might be a promising candidate for combating chemo-resistance and has the potential to
