Summary and conclusion 2013

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1 The work presented in the thesis is focused on the problems related to the prostate gland. Benign prostatic hyperplasia (BPH) and prostate cancer (PCa) are the two major problems associated with prostate. Both are age associated problems affecting majority of male of age 40 years and above. Prostate growth is normally regulated by activation of the androgen receptor (AR), a ligandactivated nuclear transcription factor. The steroid enzyme 5α-reductase converts testosterone into dihydrotestosterone (DHT), an active androgen, which binds to AR leading to its nuclear translocation for the transcriptional activation of androgen-responsive genes. Prostate specific antigen (PSA) and AR target gene, is thought to contribute to PCa progression. Although metastatic PCa remains an incurable disease at present, therapy can only delay progression. The first step in treatment of metastatic disease is to block testosterone-driven proliferation of PCa cells through androgen deprivation therapy (ADT) or surgical castration. Recent studies have suggested that ADT do not cause complete depletion of androgens. In such conditions, even nanomolar concentrations of testosterone and DHT, most likely produced by adrenal glands or tumor itself, are sufficient for the transactivation of AR to support PCa cell proliferation, furthermore ADT causes adverse effects like hypogonadism which leads to increase in body mass index, declines in lean body mass, muscle strength, bone density, sexual function and quality of life. PCa requires at least years to develop and express its clinical symptoms. Many autopsy cases have confirmed presence of initial stages of prostate cancer but the death of the individual was due to other reasons, with increase in age this could be a serious problem. Hence it presents a unique challenge because of its long latency, high prevalence, screening complexity and significant mortality and morbidity making it an important target for chemoprevention. Therefore it provides an ideal window of opportunity for chemopreventive intervention. Whereas, it is likely that chemoprevention modality of cancer management from pre-neoplastic conditions to high-grade prostatic intraepithelial neoplasia (PIN) is not necessarily eliminate but, it is expected to halt neoplasticity. Suppression of progression of preneoplastic lesions by use of chemopreventive agents certainly would improve morbidity and survival time in PCa patients. The anti-cancer efficacy of chemopreventive agents has been linked to the modulation of Jamia Hamdard, Department of Medical Elementology and Toxicology 119

2 mitogenic signaling, cell-cycle regulation, survival/apoptotic signalling, angiogenic, and metastatic events in cancer cells of PCa. Chemopreventive agents also possess anti-oxidant activity and can inhibit inflammation and stimulate phase II detoxification enzyme activity. International variations in cancer rates have been attributed, at least in part, to differences in dietary intake. Recently, it has been reported that there was a decrease in prostate cancer incidences in Asian countries when compared to their western counter parts, to some extent it was associated to high intake of soy foods in their diet. Epidemiologic and laboratory evidence support the hypothesis that PCa risk is the result of combinatorial impacts of crucial environmental exposures, inherited susceptibility, and modifying influences of diet and lifestyle factors. Flavonoids are nonessential dietary factors, and humans consume about 1-2 g of flavonoids daily. Flavonoids are abundantly present in fruits, vegetables, seeds, nuts, tea, and red wine. Flavonoids are believed to act as health-promoting substances, and some of them have antioxidant and anti-inflammatory properties. Flavonoids are free radical scavengers, they protect organisms from carcinogenic reactive oxygen species (ROS) and other radicals. Flavonoids are known to display anti-inflammatory capacities, an important feature because inflammatory processes are involved in cancer development, especially when they are longlasting or excessive and destroy epithelial barriers. Taking into consideration the above facts, the present work was carried out to minimize the drug induced side effect in prostate associated diseases and to understand the mechanism associated with prostate proliferation, BPH and PCa. Our other aim was intercepting the disease and prolong the progression phase by use of dietary interventions. As chemoprevention is the better method of choice keeping in view the long latency of PCa, two flavonoids daidzein belonging to isoflavon sub-class and luteolin belonging to flavone sub-class were selected. Both the compounds have been reported to have antioxidant, anti-inflammatory, anti-proliferative properties. It is well established fact that androgens and AR govern the growth and differentiation of prostate gland/prostate cancer. Hence androgen deprivation therapy is the treatment of choice for prostate problems (BPH and PCa). Secondly many researchers reported the increase in number of mortalities of PCa patients who were on long term androgen deprivation therapy due Jamia Hamdard, Department of Medical Elementology and Toxicology 120

3 to cardiovascular diseases. So, the first experiment (chapter III) was done to study the effect of sub-chronic androgen deprivation on the antioxidant status, inflammation and extracellular matrix degradation. We used flutamide a known non-steroidal anti-androgen drug to induce androgen deprivation in wistar rats. Subchronic androgen deprivation alleviated oxidative stress, inflammation and degradation of extracellular matrix degrading proteases (upar and MMP-9). Androgen deprivation also led to altered lipid profile. Daidzein and luteolin administration maintained the extracellular matrix proteases homeostasis, restored the antioxidant enzyme levels and suppressed inflammation. Only daidzein was able to normalize the lipid profile, where as luteolin did not show any significant change to lipid profile. The second most common problem of prostate which affects majority of the males above 40 years is BPH. It is a well-known condition characterised by prostate growth accompanied by lower urinary tract symptoms. BPH is a proliferative disease of the secretory and stromal cells. Prostate is a mature gland with fully developed immune system. The pathogenesis of BPH is not yet fully understood. There are several mechanisms involved in the development and progression of the disease like age-related tissue modifications, hormonal alterations, and metabolic syndromes as well as inflammation are some of them. Many clinical and basic research papers support the hypothesis that BPH predominantly consists of an immune inflammatory response of prostatic tissue to an unknown stimulus that would create a proinflammatory milieu within the gland. This theory is confirmed by several basic research and clinical studies that showed a statistically significant association between inflammation and BPH severity and progression. Therefore, from the recent clinical and basic research studies, a novel approach in the clinical management of BPH might focus on the inflammatory process involved in the development and progression of the disease. Many researchers developed BPH in rodents by repeatedly administration of testosterone hormone. Since inflammation is one of the main reasons for prostate hyperplasia, the use of anti-inflammatory agents may be an ideal choice to keep a check on BPH. Flavonoids are well reported to possess anti-oxidant and antiinflammatory properties. Moreover With increase age there is gradual alleviation in the oxidative stress, resulting in increased inflammation which is proposed to be one of the causes of prostate secretory and stromal cell proliferation. Our aim was to decrease the inflammation and oxidative stress by use of dietary agents. Jamia Hamdard, Department of Medical Elementology and Toxicology 121

4 We induced BPH by repeated high dose of testosterone propionate in rats. We studied the antioxidant enzyme levels and the levels of inflammatory and anti-inflammatory cytokines. The prophylactic treatment of rats with daidzein and luteolin resulted in decreased activation of T- cells which are responsible for the production of inflammatory cytokines. The was a decrease in Mast cell infiltration which recruiters inflammation cytokines by daidzein and luteolin administration. We also found restoration in the antioxidant enzymes, suppressed inflammatory cytokines IL 1β, IL 6, TNF α, IFN γ levels and attenuated the activation of NFκB, COX-2 production. This may have led to the decrease in prostate hyperplasia. (Chapter IV) Prostate cancer is second leading cancer in the world. Globally the incidences of prostate cancer are increased at geometric rate, paradoxically the advancement in therapy at the arithmetic rate. Androgen deprivation is the sole method of choice to keep a check on the prostate cell proliferation and induce cell death in the prostate cancer cells. On the contrary long term androgen deprivation leads to the onset of chronic refractive prostate cancer which by any means does not respond to androgen deprivation therapy. Modern science fraternity comes to stand still after the emergence of this cancer stage and the clinicians are rendered helpless. Presently we do not have a gold standard therapy where in we can prevent the onset of this condition. It is well establish fact the PCa has long latency period i.e approximately 30 years. This is the most critical period where in we can intercept the disease and delay its onset. Modern days the emergence of naturopathies having minimal toxicities has emerged as the preferred choice of therapy. Our aim was to intercept the disease at promotion phase and increase its duration. Secondly to understand the mechanism of action of daidzein and luteolin in chemically induced two stage prostate carcinogenesis in Wistar rats. In this study two flavoids daidzein and luteolin were used to intercept the disease. We found a decrease in inflammation which is one of the important factors in cell proliferation. We found a decrease inflammatory cytokines (IL 1β, IL 6, TNF α, IFN γ), decreased mast cells infiltration and inhibited COX-2 secreation. NFκB the master of inflammatory orchestra was also down regulated by daidzein and luteolin administration. Daidzein and luteolin also restored the antioxidant enzymes, induces cell death in the DNA damaged cells. This is clearly evident by the Jamia Hamdard, Department of Medical Elementology and Toxicology 122

5 activation of p53/p21 expression and induction of caspases, it was further confirmed by BAX/BCl 2 ration which was greater than one (>1) (chapter v) When we compare both the modulators used throughout the work we can say the daidzein proved to be better than luteolin. It maintained lipid profile, ECM homeostasis and oxidative stress in ADT rats better then luteolin. In case of BPH also daidzein was more potent in retarding prostate proliferation, inflammation as compared to luteolin. When it comes to chemoprevention of prostate cancer, daidzein proved to be better by higher decrease in PSA, testosterone and DHT levels and it not only decreased in the inflammation in prostate cells but also induced cell death (caspase activation and BAX/Bcl 2 ) better then luteolin. Finally to conclude we can say that daidzein and luteolin proved to be better agents when can by incorporated as dietary agents to minimize the toxicities associated with prostate cancer drugs; may help in better management of patient who are on long-term androgen deprivation therapy, to prevent the onset of BPH and more importantly when incorporated as dietary agent there are very much probability that there may be a delayed onset in prostate cancer. More work is needed to understand the mechanism of action of these two modulators, so that they can be harnessed for the better management of prostate cancer patients. Jamia Hamdard, Department of Medical Elementology and Toxicology 123

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