Effect Of Lycorine Hydrochloride On Prostate Cancer And Mechanism Study

BackgroundProstate cancer (Pca) is a kind of malignancy occurred in prostatic epithelium and it is a common malignant tumor in old male. In the worldwide range, the morbidity of prostate cancer ranks the second of all male malignant tumors and the mortality in the sixth, so it is seriously threatening human health. In the past, the morbidity of prostate cancer is relatively low. But with the aggravation of aging population, pollution of living environment, change of eating habits and gradual popularization of prostate specific antigen in recent years, its morbidity is gradually on a rise. According to different disease conditions, the treatment ways of prostatic cancer include active surveillance, radical operation, external beam radiotherapy (EBRT), close irradiation treatment, local experimental prostate cancer treatment and endocrine therapy, etc.Currently, endocrine therapy is one of the standard treatment means for early Prostate Cancer. But on clinical, it was found that after being treated by endocrine therapy for a period of time, most patients developed into Castration-Resistant Prostate Cancer (CRPC) and became insensitive to androgen blockade and the malignant degree of tumors was high.Chemotherapy can be used to treat Castration-Resistant Prostate Cancer. Currently, chemotherapy with docetaxel as the basis has become a standard first-line chemotherapy for this kind of patients. But most chemotherapy drugs have shortcoming like, relatively large toxic side effects, poor selectivity or multidrug resistance. In recent years, newly-developing drugs like Abiraterone Acetate were proven to have a certain curative effect for late stage of prostate cancer, but the effective rate of these drugs are not high and they are also very expensive, which greatly limited their clinical application in our country. Sipuleucel-T (Provenge) is the first effective tumor vaccine that is used for treating Castration-Resistant Prostate Cancer, but it also makes patients face the problem of high treatment cost. Therefore, it is very necessary to research and develop some safe, effective and inexpensive drugs to treat prostatic cancer.At present, in the aspect of developing antitumor new drugs, to extract effective anti-cancer ingredient from natural plants has become an important research direction. During the development process of anti-tumor natural drugs, alkaloid is a major branch of present studies. Alkaloid is a kind of alkaline organic compound containing nitrogen and it has the chemical property similar to alkaline, mainly existing in the animals and plants in nature. Most alkaloids have complex cyclic structures and nitrogen is contained in the cycle. The structure based this presents rich physiological and pharmacological activities. Currently, over 2000 kinds of alkaloid have been explored. In the biomedical field, alkaloids are divided into seven types according to their chemical structures:pyridines and piperidines, organic amines, indoles, tropane, steroids, isoquinolines and terpenes.Lycorine belongs to isoquinoline alkaloids and is the derivative of pyrrolo-phenanthridine. Lycorine is the first alkaloid that is separate from the amaryllidaceae plant and is also the most common alkaloid in this kind of plants. Lycorine has a variety of bio-pharmacology activity. The current study proves that lycorine has the effect of anti-tumor, anti-virus, anti-inflammatory, anti-malaria, inhibiting acetylcholinesterase activity, inhibiting vitamin biosynthesis and protecting angiocarpy. In recent years, the rich pharmacology and physiological activity of lycorine has caused the wide attention in the field of biomedical research and there are more and more reports about the anti-tumor effect and mechanism of lycorine and its derivatives. Until now, a lots of studies have proved that the growth inhibition effect of lycorine on tumor cells is significantly higher than its growth inhibition effect on normal cells. The effect of lycorine on tumors shows a broad spectrum. The current studies revealed that lycorine and its derivatives have different degrees of inhibiting effect on leukemia, lymphoma, melanoma, esophageal cancer, breast cancer, ovarian cancer, prostate cancer and other tumors. The previous studies revealed the complexity of the anti-tumor effect of lycorine. Whether there are other mechanisms on the skilling effects of lycorine on prostate cancer cells is still unknown. Therefore, it is necessary to explore the influence of lycorine on prostate cancer cells and explore the possible new mechanisms.NF-kB is a kind of protein complex that control DNA transcription, cytokines generation and cell survival. NF-kB almost exists in all types of animal cells and participates in responses of cells to many stimuli, which include stress, cell factors, free radicals, ultraviolet radiation, oxidation LDL and bacteria or virus antigen. In mammals, NF-kB family consists of five related transcription factors:P50/NF-kB1, P52/NF-kB2, REL/c-Rel, Rel-A/p65 and Rel-B. These transcription factors all have an N-terminal DNA dimerization domain that is consisted by 300 amino acids, called Rel homology domain (RHD). Through RHD, NF-kB family members can form homologous and heterodimer. Rel A, c-Rel and RelB gene contains c-terminus transcription activating domains, which can make them activate the expression of target expression. On the contrary, p50 and p52 do not contain c-terminus TADS; therefore, p50 and p52 dimers inhibit transcription, unless they combine with proteins containing TADS, such as RelA, c-Rel, RelB or BCL-3 (one transcription-related coactivator).The transcription activity of NF-κB was inhibited by IκB protein existing in cytoplasm. Currently, there are seven defined IκB family member, including IκBα IκBβ, Bcl-3, IκBε, IκBγ and precursor protein P100 and P105. When NF-kB was combining with IκB, NF-κB cannot enter the nuclear, so it does not have transcription activity. After IκB was phosphorylated, NF-κB can relieve the inhibition effect of IκB and enter into the cell nuclear and adjust the transcription of related target genes. A large number of cytokines, carcinogenic agents, and tumor microenvironment can activate the NF-κB through relative signal pathway, so as to regulate the expression of downstream protein, involve in the occurrence, development, invading and transferring of tumors. It has been confirmed by studies that NF-κB is in abnormal activating state in many malignant tumors, while inhibiting NF-κB activity can inhibit the development of various tumors, indicating that NF-κB plays an important role in the occurrence and development of tumors. NF-κB signal pathway is closely related to prostatic cancer. Many studies porved that, NF-κB was continusouly active in a series of prostatic cancer cell lines and prostatic cancer animal models and NF-kB high activation was found in prostatic cancer cell lines PC-3, DU145 lack of androgen receptor expression.Inhibitor of apoptosis proteins (IAPs) is a kind of highly conservative endogenous anti apoptosis factor family and they mainly inhibit cell apoptosis by inhibiting Caspase activity and involving regulating nuclear NF-κB effect. Survivin is a member of the IAP family, with the effect of anti-apoptosis, regulating cell cycle and involving in angiogenesis. There is rarely no expression of Survivin in the normal matured tissue (except the thymus gland and the gonads) while there is a high expression in most common malignant tumors. American Institute for Cancer Research detected 60 kinds of tumor cell lines and the results showed that there was a high expression of Survivin in 60 kinds of tumor cell lines, including kidney cancer, prostate cancer, breast cancer, liver cancer, colon cancer, melanoma, lymphoma, leukemia, etc. From above, we can conclude that Survivin was closely related to urinary tract tumors. The study found that there was expression of Survivin in different prostate cancer cell lines, indicating that Survivin plays an important role in the occurrence and development of prostate tumors. The study also found that Survivin was not only highly expression in prostatic cancer, but was also related to the clinical staging and pathology grading of prostatic cancer.To sum up, there is an upward trend in prostate cancer incidence; NF-κB signal pathway is an important signal pathway in prostate cancer; Lycorine has showed a good anti-tumor activity. At present, there is no research that explores the influence of LH on prostate cancer cells through NF-κB signal pathway and its downstream Survivin. Therefore, we think that it is necessary to make clear the effect of LH on prostate cancer cells. First, this study has discussed the influence of LH on NF-κB signal pathway in prostate cancer cells, then explored the effect of LH on downstream target Survivin of NF-κB signal pathway and finally analyzed the influence on LH on prostate cancer cell phenotype through experiments on cell proliferation, cycle, apoptosis, tumor clone formation, tumor cell migration, nude mice model bearing subcutaneous tumor, etc. This paper can be divided into the following three chapters.Chapter One Lycorine Hydrochloride Inhibiting NF-κB Signal Pathway of Prostate Cancer CellsObjectives:lycorine hydrochloride plays the role of tumor suppressor in a wide variety of tumors. But there are no studies exploring the effect of lycorine hydrochloride on NF-κB signal pathway of prostate cancer cells. This study is to explore the influence of lycorine hydrochloride on NF-κB signal pathway in PC-3 and 22Rvl cells of prostate cancer cell strain.Methods:PC-3 and 22Rv cells were acted on by LH, BAY and LH+BAY for 48 hours, respectively and 40ng/ml TNF-α was used to stimulate NF-κB signal pathway in PC-3 and 22Rv1 cell lines. The total cell protein was extracted at Omin and 15min and Western Blot was adopted to detect the expression condition of NF-κB signal pathway associated protein P-IKKα/β, IKKα/β, P-P65, P65, P-IκBα and IκBα in PC-3 and 22Rv1 cells.Results:compared with DMSO group, after being acted on by LH, BAY and LH +BAY, P-IKKα/β, P-P65, P-IκBα protein expression amount in PC-3 and 22Rv1 cells evidently decreased.Conclusions:lycorine hydrochloride can efficiently inhibit NF-κB signal pathway in PC-3 and 22Rv1 cell lines of prostate cancer.Chapter Two Lycorine Hydrochloride Inhibiting Survivin Expression in Prostate Cancer CellsObjectives:This study is to explore the influence of lycorine hydrochloride on the downstream target surviving of NF-κB signal pathway in prostate cancer cells.Methods:PC-3 cells were acted on by 2μg/ml LH,5μM BAY, 10nM YM-155, 2μg/ml LH+5μM BAY,2μg/ml LH+lOnM YM-155,5μM BAY+10nM YM-155, 2μg/ml LH+5μM BAY+10nM YM-155 for 48h, respectively. The total RNA and total protein were extracted respectively.qPRC, Western Blot and fluorescence detection were separately adopted to detect the influence of LH on survivin mRNA and protein expression level in PC-3 cells.Results:Compared with DMSO control group, after being treated by 2μg/ml LH,5μM BAY, lOnM YM-155,2μg/ml LH+5μM BAY,2μg/ml LH+lOnM YM-155,5μM BAY+lOnM YM-155,2μg/ml LH+5μM BAY+10nM YM-155, mRNA and protein level of surviving expression in PC-3 cells both evidently decreased (P<0.05). The immunofluorescence results indicated that:after being treated by DMSO, surviving presented strong fluorescence staining in prostate cancer PC-3 cells; while compared with DMSO group, after being treated 2μg/ml LH,5μM BAY, 10nM YM-155,2μg/ml LH+5μM BAY,2μg/ml LH+10nM YM-155, 5μMBAY+10nM YM-155,2μg/mlLH+5μMBAY+10nM YM-155, the fluorescent staining in PC-3 cells gradually weakened.Conclusion:lycorine hydrochloride can effectively inhibit the activity of downstream target surviving of NF-kB signal pathway in prostate cancer cell lines and this kind of inhabiting effect has synergistic effect when combined with BAY, YM-155.Chapter Three The Influence of Lycorine Hydrochloride on Prostate Cancer Cell PhenotypeObjectives:this study is to explore the influence of lycorine hydrochloride on biological characteristics of PC-3 and 22Rvl cells of prostate cancer cell lines.Methods:prostate cell lines PC-3 and 22Rvl cells were taken as the study objects and the experimental treatment groups were set as:(1) DMSO treatment group; (2) LH treatment group; (3) BAY treatment group; (4) YM-155 treatment group; (5) LH+BAY combined treatment group (L+B); (6) LH+YM-155 combined treatment group (L+Y); (7)BAY+YM-155 combined treatment group(B+Y); (8) LH+BAY+YM-155 combined treatment group (L+B+Y). PC-3 and 22Rvl cells were acted on for 24-48 hours. Then MTS was adopted to study the influence of LH on proliferation of prostate cancer cells in vitro; flow cytometry was adopted to study the influence of LH on prostate cancer cell apoptosis and cycle; plate clone formation was performed to study the influence of LH on colony forming ability of prostate cancer cells in vitro; Transwell was performed to study the influence of LH on transfer ability of prostate cancer cells in vitro; at the same time, nude-mouse transplanted tumor model was established to study the anti-prostate cancer effect of LH in vivo.Results:1.2,3,4,5 d after intervention of LH, it significantly inhibited proliferation of PC-3 and 22Rvl cells (P<0.05) and its proliferation inhibitory degree on PC-3 and 22Rvl cells was almost the same with the inhibitory degree of BAY and YM-155 (P>0.05). At the same time, when LH was combined with BAY or YM-155, LH can strengthen the inhibition effect of BAY and YM-155 on PC-3 and 22Rvl cell proliferation.2. The influence of LH on prostate cancer cell apoptosis:LH can significantly promote the apoptosis of PC-3 and 22Rvl cells (p< 0.05). Its induced apoptosis degree on PC-3 and 22Rvl was quite the same with BAY and YM-155 (P>0.05). But when LH was combined with BAY and YM-155, it can strengthen the promoting effect of BAY and YM-155 on PC-3 and 22Rvl cell apoptosis.3. The influence of LH on prostate cancer cell cycle:LH can significantly reduce the percentage of S phase cells in PC-3 and 22 Rvl (p< 0.05). Its effect was similar to BAY and YM-155 (P>0.05). But when LH was combined with BAY and YM-155, it can strengthen the reducing effect of BAY and YM-155 on percentage of S phase cells in 22 Rvl (p< 0.05).4. The influence of LH on in vitro cloning formation ability of prostate cancer cells:compared with DMSO treatment group, the cell clone formation ability of LH treatment groups significantly decreased (p<0.05), while there was no difference between LH and BAY treatment group or LH and YM-155 treatment group (p>0.05). Meanwhile, compared with single drug treatment group, a combination of two drugs or three drugs can evidently reduce the cloning formation ability of PC-3 and 22 Rvl, indicating that LH can inhibit the in vitro cloning formation ability of prostate cancer cells.5. The influence of LH on transfer ability of prostate cancer cells in vitro: compared with the control group, after being treated by 2μg/ml LH,5μM BAY, 10nM YM-155,2μg/ml LH+5μM BAY,2μg/ml LH+10nM YM-155,5μM BAY+lOnM YM-155,2μg/ml LH+5μM BAY+lOnM YM-155, the number of PC-3 cells and 22Rvl cells that can pass through chamber both decreased evidently (p< 0.05); Compared with the single drug treatment group, the number of PC-3 cells in a combination group of two drugs or three drugs that can pass through chamber both decreased evidently (p<0.05). There was no difference among single drug groups of LH, BAY and YM-155 (P>0.05) and there was no difference among L+B, L+Y and B+Y treatment groups (p>0.05). It indicated that LH can inhibit the transfer ability of prostate cancer cells. Meanwhile, a combination of LH and BAY and YM-155 can strengthen the inhibiting effect on transfer ability of prostate cancer cells.6. The influence of LH on subcutaneous tumor formation ability of prostate cancer cells. Compared with PBS control group, LH and YM-155 can both significantly inhibit the growing rate and tumor size of transplant tumor (p<0.05), and there was no difference between LH injection group and YM-155 injection group (p>0.05). Meanwhile, compared with PBS control group, LH and YM-155 can also significantly inhibit the weight of tumor (p<0.05).Conclusion:LH can inhibit the proliferation of prostate cancer cells by inhibiting NF-kB and its downstream target Survivin, promote apoptosis, block the cell cycle, reduce the cell migration ability, reduce in vivo tumorigenesis, and the inhibition effect has synergistic effect when LH is combined with BAY, YM-155.