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Advanced prostate cancer has significant long-term morbidity, and there is a growing interest in alternative and complimentary forms of therapy that will improve the outcomes of patients. Azadirachta indica common name: neem contains multiple active compounds that have potent anti-inflammatory and anticancer properties. The present study investigates the novel targets of the anticancer activity of ethanol extract of neem leaves EENL in vitro and evaluates the in vivo efficacy in the prostate cancer models.
Genome-wide expression profiling, using oligonucleotide microarrays, Prostatakrebsbehandlung Innovation genes differentially expressed with EENL treatment in prostate cancer cells.
Functional analysis unveiled that most of the up-regulated genes were associated Prostatakrebsbehandlung Innovation cell death, and drug metabolism, and the down-regulated genes were associated with cell cycle, DNA replication, recombination, and repair functions.
The suppression of tumor growth is associated with the formation of hyalinized fibrous tumor tissue and the induction of cell death by apoptosis. These results suggest that EENL-containing natural bioactive compounds could have potent anticancer property and the regulation of multiple cellular pathways could exert pleiotrophic effects in prevention and treatment of prostate cancer. The online version of this article doi Prostate cancer is one of the most prevalent malignant neoplasms and the third leading cause of cancer-related death of men of the Western countries 1.
The mainstay of treatment of advanced prostate cancer is focused on suppression of intraprostatic testosterone and dihydrotestosterone DHT actions 2.
However, after an initial Prostatakrebsbehandlung Innovation, therapy-resistant clones can appear and result in cancer progression and metastasis with high mortality 3. First-line chemotherapy for advanced prostate cancer has not demonstrated significant improvement in overall survival but could provide disease control and palliation 4. Novel treatment modalities are Prostatakrebsbehandlung Innovation needed to treat hormone-resistant tumors and to prevent the progression of hormone-sensitive prostate cancer to hormone-refractory stage.
The search for compounds with few or no adverse effects that will prevent cancer progression and protect against the adverse biological effects of chemotherapeutic agents as compared with the agents currently in use is therefore of greatest relevance. Herbal plants and plant-derived medicines have been used as the source of potential anticancer agents in traditional cultures all over the world and are becoming increasingly popular in modern society 5.
The potential natural product-derived anticancer agents are known to possess various bioactive phytochemicals. Terpenoids constitute one of the largest families of natural products accounting for more Prostatakrebsbehandlung Innovation 40, individual compounds of both primary and secondary metabolisms 6. Many herbal plants contain terpenoids, and several terpenoids have been shown to be available for pharmaceutical applications, for example, artemisinin and taxol as malaria and cancer medicines, respectively 7.
Neem is one such medicinal plant, the extract of which has been used for thousands of Prostatakrebsbehandlung Innovation for most acute and chronic diseases in India and Africa. The major biologically active constituents of neem leaves are limonoids, triterpenoids, nonterpenoids, phenolics, flavonoids, and meliacins 89potentially targeting multiple signaling pathways Prostatakrebsbehandlung Innovation cancer cells 10 — Extract of neem leaves have been reported to be non-toxic, non-mutagenic, and found to possess immunomodulatory, anti-inflammatory, and anticarcinogenic properties 13 Neem leaf Prostatakrebsbehandlung Innovation exhibited antitumor activity by activation of cytotoxic Prostatakrebsbehandlung Innovation lymphocytes and natural killer Prostatakrebsbehandlung Innovation in patients with head and neck squamous cell carcinoma To date, there is only one report of neem on prostate cancer which showed the in vitro inhibition of PC-3 cell proliferation and Bcl-2 expression after neem treatment No further studies of any neem compounds or extracts were reported on prostate cancer.
The above reasoning promoted use Prostatakrebsbehandlung Innovation explore the antitumor effects of Prostatakrebsbehandlung Innovation leaves on human prostate cancer cells which could lead to Prostatakrebsbehandlung Innovation clinical trials for Prostatakrebsbehandlung Innovation cancer patients. Our study is designed to identify and evaluate the molecular targets of anticancer activities of ethanol extract of neem leaves EENL in prostate cancer Prostatakrebsbehandlung Innovation.
To unravel the molecular effects of EENL in androgen-refractory metastatic prostate cancer cells, we used gene expression microarrays and identified target genes regulated Prostatakrebsbehandlung Innovation prostate cancer cells after treatment with EENL. We then confirmed the alterations in mRNA and protein Prostatakrebsbehandlung Innovation levels of the genes.
The residue was freeze-dried and yielded approximately 1. The suspension was filtered using a 0. The stock solution was further diluted with ethanol for all the experiments. The final concentration of DMSO in the culture medium never exceeded 0. The effect of the extract on cell viability and gene expression levels described below were assessed to standardize the method of extraction.
We obtained consistent results with different lots of the extract. High-performance liquid chromatography HPLC -grade acetonitrile, water, isopropanol, and methanol were purchased from Burdick and Jackson Muskegon, Michigan.
Louis, Missouri. The makeup of the LC mobile phases was as follows: mobile phase A Prostatakrebsbehandlung Innovation acid The flow rate was 0. PC-3M-luc2, originated from PC3, is a luciferase-expressing Prostatakrebsbehandlung Innovation prostate cancer cell line which was stably transfected with firefly luciferase gene luc2was purchased from Caliper LifeScience Hopkinton, MA.
CB cells were treated with 5. CB cells were treated with 8. These results were then separated by significant up-regulated or down-regulated genes, Prostatakrebsbehandlung Innovation used for further validation. The cDNA isolated as described above was mixed with the Taqman universal master mix and loaded Prostatakrebsbehandlung Innovation to the microfluidic cards.
GAPDH was used as loading control. The animals Prostatakrebsbehandlung Innovation with CB and PC-3M-luc2 cells were randomly assigned to three groups of six each and two groups of six each, respectively. Animals were weighed once every week to monitor the effect of EENL toxicity on body weight. In brief, prostate tumor tissues were thawed, weighed, and homogenized in 1. This was followed by conventional LC on a multiplexed liquid chromatography system and analyzed on a tandem mass spectrometer equipped with an electrospray interface.
Fifty percent inhibition concentration IC 50 values Prostatakrebsbehandlung Innovation calculated by Probit Prostatakrebsbehandlung Innovation. Partek Genomics suite 6. The total ion chromatogram of the EENL depicts seven significant, based on intensity, peaks.
Peak 3 has a retention time of This is possibly suggestive by mass alone as nimolinone. This is possibly suggestive by Prostatakrebsbehandlung Innovation alone as 6-desacetyl nimbinene. In an initial approach Prostatakrebsbehandlung Innovation analyze the potential of whole EENL for anticancer activity, we performed viability assays employing 2 frequently used human androgen-refractory CB and PC-3M-luc2 prostate cancer cell lines.
Vehicle-treated cells were included as a control. Inhibition of prostate cancer cell proliferation by treatment with ethanol extract of neem leaves EENL. To identify the molecular targets of the anticancer effects of EENL, we performed high-resolution whole genome profiling using an Affymetrix microarray platform. Using the Ingenuity Pathways Knowledge Prostatakrebsbehandlung Innovation, the dataset was used to map independently up- and down-regulated genes for the molecular and cellular functions.
The Prostatakrebsbehandlung Innovation enriched functions of Prostatakrebsbehandlung Innovation up-regulated genes are Prostatakrebsbehandlung Innovation death, cellular development, cellular growth and proliferation, lipid metabolism, and small molecule biochemistry. The majority of the up-regulated genes are associated with cell death function which Prostatakrebsbehandlung Innovation that EENL could play a vital role in promoting cell death.
The most significant down-regulated gene functions are Prostatakrebsbehandlung Innovation cycle, DNA replication, recombination, Prostatakrebsbehandlung Innovation repair, cellular assembly and organization, cellular movement, and gene expression. To validate the observed changes in gene expression, we chose 40 Prostatakrebsbehandlung Innovation genes Prostatakrebsbehandlung Innovation Taqman real-time PCR analysis and analyzed in both CB and PC-3M-luc2 prostate cancer cells.
For all genes tested, the direction of gene expression change measured by RT-PCR and microarray analysis agreed, although the magnitude of expression change was not always the same using these 2 different analytical methods. These results support the findings obtained from the microarray experiments. Though we only validated up-regulated genes, we believe these results can support the validity of down-regulated genes of our microarray data.
We selected four significantly up-regulated genes to confirm the protein expression by Western Prostatakrebsbehandlung Innovation analysis. Vehicle-treated cells were used as control. The experiments were repeated thrice and the representative blot was shown. To evaluate the antitumor efficacy of EENL in vivowe used xenograft tumor models. Tumor growth was significantly inhibited in all the EENL-treated groups. These findings elucidate that EENL can suppress tumor growth.
There was no significant change in body weight in any of the groups after treatment which suggests that current EENL extract causes no major toxicity to mice Fig. Tumors were assessed histologically for fibrosis, coagulative tumor necrosis, and apoptosis. Mice treated with EENL showed greater degree of fibrosis and increased apoptotic activity, whereas vehicle-treated group exhibited greater amounts of coagulative tumor necrosis Fig.
Further, we demonstrated the presence of apoptotic cells in the tumor tissue by ApopTag peroxidase staining. Prostatakrebsbehandlung Innovation results indicate that the possible mechanism for regression is by inducing apoptosis of the tumor cells. Two sections of tumor tissue from each mouse and six mice in a group were examined for histological changes.
Detection of apoptotic cells in Prostatakrebsbehandlung Innovation tumor tissue. ApopTag peroxidase in situ oligo ligation apoptosis detection kit was used for staining apoptotic cells in the tumor tissue at Prostatakrebsbehandlung Innovation end of treatment. In advanced castration recurrent prostate cancer, multiple aberrant pathways can be potentially targeted for the therapeutic effect that would yield Prostatakrebsbehandlung Innovation outcomes than monotherapies Neem contains multiple active compounds that work simultaneously via different mechanisms 10 — The scientific evaluation of neem as an anticancer agent is largely unknown.
We used EENL extract to evaluate the anticancer activity. In this study, we evaluated the anticancer effects of EENL in castration-resistant CB and highly metastatic PC-3M-luc2 prostate cancer cells with an intent to use the neem extract for locally advanced and metastatic prostate cancer that is associated with considerable morbidity and mortality.
The extent of cell growth inhibition was measured Prostatakrebsbehandlung Innovation MTS assay which was used to determine the number of viable cells in proliferation.
To unravel Prostatakrebsbehandlung Innovation molecular targets involved in mediating Prostatakrebsbehandlung Innovation effect of EENL on prostate cancer cells, we used genome-wide microarray analysis. PCR results were consistent with the microarray data. Most of these up-regulated genes have been previously shown to be down-regulated in human prostate cancer tissues using microarray analysis 1823 — The RNA expression profiles of the 40 most significantly down-regulated genes were shown from our microarray analysis Supplementary Table S 1.
All these 40 down-regulated genes were found to be up-regulated in various cancer tissues as shown in the Oncomine microarray data base HMOX1the inducible isoform is a rate-limiting enzyme Prostatakrebsbehandlung Innovation heme degradation 28 Numerous studies have focused on the Prostatakrebsbehandlung Innovation ablation, by Prostatakrebsbehandlung Innovation testosterone synthesis and blockade of androgen receptor, as the major treatment for hormone-sensitive prostate cancer 35 Steady-state levels of intracellular DHT are maintained through a balance between local synthetic and catabolic rates.
However, little emphasis has been placed on the importance Prostatakrebsbehandlung Innovation DHT catabolism in the prostate. AKRs are phase I drug-metabolizing enzymes for a variety of carbonyl-containing Prostatakrebsbehandlung Innovation Compared to the paired benign tissues, prostate cancer tissues showed a reduced metabolism Prostatakrebsbehandlung Innovation DHT which corresponded with a loss of AKR1C2 expression Thus, Prostatakrebsbehandlung Innovation catabolism can indirectly regulate the activity of AR and thereby provides new therapeutic targets for the treatment of prostate cancer.
The over-expression of AKR1B10 was reported in early stages of well and moderately differentiated tumors and down-regulation in advanced tumor-stages with low grade of differentiation, implicating that AKR1B10 may be a helpful marker for differentiation