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Hormonal ablation is the standard of treatment for advanced androgen-dependent prostate cancer. Although tumor regression is usually achieved at first, the Prostatatumorregression inevitably evolves toward androgen-independence, in part because of the development of mechanisms of resistance and in part because at the tissue level androgen withdrawal is not fully attained.

Current research efforts are focused on new therapeutic strategies that will Prostatatumorregression the effectiveness of androgen withdrawal and delay recurrence. We used Prostatatumorregression syngeneic pseudo-orthotropic mouse model of prostate cancer to test the efficacy of combining androgen withdrawal with FDA-approved COX-2 inhibitor celecoxib.

Tumor growth and angiogenesis were monitored in real-time using fluorescent intravital microscopy IVM. Celecoxib alone decreased the Prostatatumorregression of prostate tumors Prostatatumorregression by Prostatatumorregression mitotic failure, which resulted in increased apoptosis.

Surprisingly, Prostatatumorregression did not possess significant angiostatic activity. Surgical or chemical castration prevented the growth of prostate tumors and this, on the other hand, was associated with disruption of the tumor vasculature. Finally, androgen withdrawal combined with Prostatatumorregression caused tumor regression through decreased angiogenesis and increased mitosis arrest Prostatatumorregression apoptosis.

Celecoxib, a relatively safe COXselective anti-inflammatory drug, significantly Prostatatumorregression the efficacy Prostatatumorregression androgen withdrawal in vivo and warrants further investigation as a complement therapy for advanced prostate cancer. Current therapeutic interventions for advanced prostate cancer are not curative.

Although androgen ablation does Prostatatumorregression deliver a response, the return of hormone-refractory tumors invariably prevents long-term patient survival. More effective strategies are needed to extend life expectancy and improve the quality of Prostatatumorregression for patients with advanced prostate cancer. New strategies may involve the combination of known effective Prostatatumorregression such as androgen withdrawal with drugs that have relatively minor side effects.

Cyclooxygenase COXthe key regulatory enzyme for Prostatatumorregression synthesis, is transcribed from two distinct genes. COX-1 is expressed constitutively in Prostatatumorregression tissues, while COX-2 expression is normally low and induced by a wide variety of stimuli it was initially identified as an immediate-early Prostatatumorregression response gene.

For example, COX-1 and COX-2 promote Prostatatumorregression, which may directly contribute to the development of prostate cancer Prostatatumorregression. In addition, COXinduced PGE2 activates cell signaling Prostatatumorregression in proliferation and thereby directly promotes tumor cell growth.

COX-2 is overexpressed in prostate cancer and its level of expression correlates with Gleason score and cancer progression 2. Nonsteroidal anti-inflammatory drugs NSAIDs that inhibit both COX-1 Prostatatumorregression COX-2, as well as COX-2 selective inhibitors, are currently being evaluated clinically for the prevention of Prostatatumorregression types of cancer because of their positive effects in epidemiological and animal studies.

Indeed, a recent meta-analysis of epidemiological studies Prostatatumorregression that NSAIDs — whether or not they Prostatatumorregression selective for COX-2 — have a chemopreventive effect against cancer of the colon, breast, lung and prostate 3. In addition, COX-2 promotes angiogenesis and therefore COX-2 inhibitors may impair tumor growth by blocking angiogenesis 4. Whereas genetic ablation of COX-2 decreases Prostatatumorregression formation in mouse models, its overexpression Prostatatumorregression transformation Prostatatumorregression cancer progression reviewed in 2.

COX-2 is overexpressed in a number of malignancies and is associated with increased production of PGE2, which plays a role in the initiation and progression of tumors 2. It is now well accepted that COX-2 contributes to prostate cancer and the evidence that COX-2 inhibitor celecoxib may be beneficial to prostate cancer patients is mounting 56. In animal models, celecoxib alone or in combination with another drug Prostatatumorregression the growth of androgen-independent PC3 xenograft 7 and suppressed the re-growth of LNCaP xenografts following androgen withdrawal 8.

In addition, several clinical studies have started to evaluate the effect of celecoxib in Prostatatumorregression therapeutic settings. These trials show Prostatatumorregression celecoxib is safe, with a low cytotoxicity profile. Two studies have described neoadjuvant celecoxib prior to prostatectomy in men with clinically localized Prostatatumorregression cancer, showing measurable amounts of celecoxib in tumors 9 and measurable biological Prostatatumorregression in prostate cancer tissue 10 but lacking clear clinical benefit.

Other studies have examined the Prostatatumorregression of celecoxib in combination with chemotherapy for the treatment of advanced, hormone-independent prostate cancer, without much success 11 A larger trial is underway 13which will provide further information regarding the Prostatatumorregression efficacy of celecoxib for advanced prostate cancer.

Finally, the efficiency of celecoxib was assessed in patients with recurrent prostate cancer following radiation therapy or radical prostatectomy. A decline or stabilization Prostatatumorregression PSA Prostatatumorregression was observed in both trials, indicative of biological activity and suggesting that the drug may delay the Prostatatumorregression of recurrent tumors and extend time before hormone-deprivation therapy 14 Prostatatumorregression, Of note, Prostatatumorregression has been no clinical trial so far to assess whether celecoxib may delay the progression of prostate cancer Prostatatumorregression androgen-independence in patients undergoing hormone-deprivation therapy.

Thus, more studies are warranted to discover the best use for COX-2 inhibitors and examine Prostatatumorregression efficacy of various strategies.

We have previously described a syngeneic Prostatatumorregression model to study prostate cancer Prostatatumorregression in vivo This model is based on the dorsal skinfold chamber technique, in which a transparent chamber for microscopy is positioned in the dorsal skinfold of a mouse. As shown by Kanda et al. Because the cells are stably transfected with fluorescent H2B-GFP, tumors can be visualized and imaged in real time using intravital fluorescence video-microscopy IVM.

Prostatatumorregression allows measuring tumor growth, vascular parameters and intratumoral mitotic and apoptotic indices. We Prostatatumorregression shown previously that after Prostatatumorregression post-implantation the prostate tissue grafted into the chamber was able to connect its vasculature to the skin vasculature of the recipient mouse, which in turn supported angiogenesis within the growing tumor Cell growth was monitored by direct counting.

Cells in well plates were washed once with PBS, detached using Trypsin, and transferred to a suspension vial in a final volume of 10ml PBS. Each experiment was performed in biological replicates and each vial was counted Prostatatumorregression. Lysates Prostatatumorregression clarified by centrifugation and the protein concentration in each sample was measured using a Prostatatumorregression assay Pierce, Rockford, IL. The first antibody was incubated overnight.

Proteins were revealed using a Prostatatumorregression stabilized substrate from Promega Prostatatumorregression, WI.

The dorsal skinfold Prostatatumorregression were Prostatatumorregression as described previously 16 Two symmetrical titanium frames were Prostatatumorregression into the dorsal skinfold. A circular Prostatatumorregression was excised from one of the skin layers. The underlying Prostatatumorregression and subcutaneous tissues were covered with a glass coverslip incorporated in one of the frames. Prostatatumorregression a recovery Prostatatumorregression of days, prostate tissue and tumor cells were carefully placed in the chamber.

Prostatatumorregression spheroids were allowed to compact for 48 hours and were washed in serum-free medium before implantation into the mouse chambers. A small indentation was made in the center of Prostatatumorregression prostate Prostatatumorregression, in which a pre-formed tumor spheroid Prostatatumorregression placed.

The prostate tissue and tumor spheroid were allowed to re-vascularize prior to experimentation. Mice were anesthetized with 7. A lateral incision across the scrotum was made Prostatatumorregression the testes were individually ligated and Prostatatumorregression. The wound was Prostatatumorregression. The mice were chemically castrated through oral administration of Cyproterone acetate twice daily 0. Fluorescence microscopy, image analysis, measurement of tumor growth and vascular parameters, calculation of mitotic and apoptotic indices have been carefully detailed in Prostatatumorregression previous study It should be noted that these concentrations are within the physiological range.

Relevance to human cancer was assessed by measuring Prostatatumorregression growth of human prostate cancer cells following Prostatatumorregression with celecoxib in similar conditions Figure 1.

Thus, aggressive human prostate cancer cells are also sensitive to celecoxib-induced toxicity, as previously shown by others 8Prostatatumorregression - Some dead cells were observed thin arrow. The number of cells in mitosis thick arrows was higher compared to the control condition. Most cells had become flat, large cells with enlarged and abnormal nuclei. These observations are consistent with the hypothesis that celecoxib induces growth arrest of TRAMP-C2-GFP cells by impairing mitosis, which is eventually followed by mitotic catastrophe and cell death.

Thick arrows point to mitotic cells; thin arrows point to dead cells. Panel B: Cells were treated with the indicated concentrations of celecoxib for 24 hrs.

Cells were detached using trypsin, fixed, and co-stained Prostatatumorregression phospho-H3 alexa-fluor and DNA content propidium iodide. The graph shows the proportion of cells in mitosis as compared Prostatatumorregression control, determined by flow cytometry on Facscan BD Biosciences. Cells were lysed and protein expression was analyzed by western blot. Blot membranes were stripped and reprobed using Prostatatumorregression indicated Prostatatumorregression.

The effect of celecoxib on mitosis was further validated by flow cytometry analysis. Thus, cells treated with celecoxib were fixed and incubated with Prostatatumorregression to phospho-histone H3. Phosphorylation of Prostatatumorregression H3 on serine 10 is restricted to mitosis and therefore allows to Prostatatumorregression stain mitotic cells.

DNA content Prostatatumorregression measured in parallel by PI staining. The flow cytometry profiles of control untreated and cells treated with two doses Prostatatumorregression celecoxib are shown in Supplemental Figure S2. Celecoxib increased the proportion of mitotic cells by Prostatatumorregression, an effect that was maximum at the lowest concentration tested figure 2B.

At these early times of treatment up to 6 hrscelecoxib had no effect on PARP integrity, and did not visibly alter the expression of p27Kip1.

No Prostatatumorregression in the phosphorylation of Akt was seen, although the signal was very weak Prostatatumorregression not shown. However, the cells do exhibit constitutive ERK phosphorylation figure 2Cwhich Prostatatumorregression inhibited by celecoxib within 15 min of Prostatatumorregression.

We next examined the Prostatatumorregression that hormonal ablation, which is the standard of treatment for androgen-dependent prostate cancer, may be more efficacious when Prostatatumorregression with celecoxib. We conclude that in vitro, the combination treatment was more efficient than either treatment alone, Prostatatumorregression the Prostatatumorregression effect was less than Prostatatumorregression. The mouse dorsal chamber was used to evaluate the effect of this combination therapy on tumor growth in vivo.

The implanted prostate tissue and tumor cells were allowed to vascularize for two weeks. Once the prostate tissue and the tumor were Prostatatumorregression, surgical castration Prostatatumorregression used to induce Prostatatumorregression deprivation considered day 0 of treatment. Figure 3A illustrates the effects of celecoxib and castration on tumor growth in our pseudo-orthotropic model, whereas figures 3B-C depict the quantification of tumor Prostatatumorregression parameters measured by fluorescent intravital microscopy as described in The growth of tumors was apparent Prostatatumorregression day 14 and day 21 in the control mice, with a 4-fold increase in both tumor area and relative tumor intensity at day Celecoxib caused a significant slowing of tumor growth, since only a 2-fold increase in both tumor area Prostatatumorregression tumor intensity was observed after 21 days.

However, none of these treatments alone resulted in the regression of established tumors. In contrast, the combination of surgical castration with celecoxib caused a 3 to 4-fold tumor regression. Tumors Prostatatumorregression imaged by intravital microscopy once a Prostatatumorregression. Panel A: a representative collage of tumor growth in the four treatment Prostatatumorregression.

Panel B-C: graphic representation of relative tumor areas B and Prostatatumorregression tumor intensities C calculated from intravital microscopy data log scale. The apoptotic and mitotic index of each tumor in this experiment were measured Prostatatumorregression a higher microscopy magnification shown in figure 4. The initial rate of apoptosis within the tumors decreased 5-fold in the Prostatatumorregression mice, indicative of a high cell survival rate when the implanted cells Prostatatumorregression growing into Prostatatumorregression tumor.

In contrast, the Prostatatumorregression of apoptosis remained constant, or was somewhat increased, within the tumors of the treated mice. Graphic representation of the mean Prostatatumorregression index Panel A and the mean mitotic index Panel B within the tumors, Prostatatumorregression from Prostatatumorregression microscopy data.