Telomerase enzyme inhibition (TEI) and cytolytic therapy in the management of androgen independent osseous metastatic prostate cancer

Abstract

Background: Recurrent prostate cancer can be osseous, androgen independent and lethal. The purpose is to discern the efficacy of synthetic small molecule telomerase enzyme inhibitors (TEI) alone or in combination with other cytotoxic therapies in controlling metastatic osseous prostate cancer.

Methods: C4-2B was pre-treated with a match or mismatch TEI for 6 weeks and then inoculated into nude mice subcutaneously or intraosseously. In a separate experiment, untreated C4-2B was injected into femur of nude mice. The mice were divided into seven systemic "combination" treatment groups of control, Ad-BSP-E1a virus, docetaxel, mismatch and match TEI. Serum PSA was followed longitudinally. Histology analyses and histomorphometry were performed. Repeated measure analysis was applied for statistical analysis and Bonferroni method was used in multiple comparisons.

Results: In the pre-treated study, the PSA of match treated cells in subcutaneous or intraosseous model was significantly lower than mismatch TEI or PBS treated group (P < 0.05). Histology revealed increased fibrosis, apoptosis and decreased PSA staining in the match TEI treated subcutaneous xenografts. In the combination treatment study, the PSA was significantly lower in single/double treatment and triple treatment than control (P < 0.05). Histology revealed that triple therapy mice had normal femur architecture. Histomorphometrics revealed that the area of femur tumor and woven bone was significantly positively correlated (P = 0.007).

Conclusions: Multiple lines of data point toward the efficacy of systemically administered telomerase inhibitors. Combining cytotoxic regimens with telomerase inhibitors could be beneficial in controlling prostate cancer. Clinical trials are warranted to explore the efficacy of TEI in prostate cancer.

Fig. 1

a: Logarithmic conversion of PSA values [ln(1 +PSA)] from subcutaneous tumor models originating from C4-2B cells pretreated in vitro with match TEI or mismatch TEI. Match TEI values were significantly lower than mismatch, P <0.0001. b: Logarithmic conversion of volumes of subcutaneous tumor model (vertical axis) originating from C4-2B cells pretreated in vitro with match TEI or mismatch TEI, versus time (horizontal axis). Tumor volumes were significantly lower for match TEI mice, than mismatch TEI mice on weeks 8, 10, 11, and 12 (P <0.05).

Fig. 2

These panels represent subcutaneous tumor of pre-treated C4-2B cells. Panels A,C,E,G are mismatch telomerase inhibitor (MM-TI) treated and panels B,D,F,H are match therapeutic drug telomerase inhibitor (M-TEI) treated. Panels A–F were taken at 200× magnification. A and B are hematoxylin and eosin stained. Panels C and D are PSA immunohistochemistry. Panel C reveals the speckled brown staining representing PSA expression throughout the tumor. Arrow indicated PSA positive cells. However, in panel D, the match treated only showed few PSA positive cells. Most of the tumor mass was negative for PSA cells. The corresponding mouse serum PSA in the match treated was also much lower, as seen in Figure 1. Panels E and F are trichrome staining, and Panel E, the mismatch treated, shows viable C4-2B cells. However, in panel F, there were more are as in blue (arrow) represent cellular debris and fibrosis than in panel E. It is apparent that much of this subcutaneous tumor mass was non-viable and filled with fibrosis. Panels G and H are TUNEL staining taken at 40× magnification. Small 200× power enlargements were embedded in the upper right corner of panels G and H, the arrows indicated the apoptotic cells. As seen in panel G, the mismatch treated, there are small brown areas of cell turnover and apoptosis, the majority of the tumor section is viable. However, in panel H, match TEI treated, most of the tumor mass has brown positive staining from TUNEL, indicative of likely non-viable cells. The subcutaneous tumors depicted herein of mismatch and match treated mice are quite representative of all the experimental specimens.

Fig. 3

a: Logarithmic conversion of PSA values [ln(1 +PSA)] from intraosseous tumor models originating from C4-2B cells treated invitro with match TEI, mismatch TEI, or PBS control. There is no statistical difference between mismatch TEI, and PBS treated groups (P-value =1.000). Match TEI treated mice had significantly lower PSA values than PBS, and mismatch TEI combined groups (P-value 0.0055). Panel b reveals all the three groups, saline control (left), mismatch TEI, and match TEI(right), with SEM bars.

Fig. 4

Femurs from PBS treated (control), mismatch telomerase inhibitor, which also is another control, and match telomerase inhibitor. Control PBS is the far left column. Mismatch telomerase inhibitor is the middle column, and match telomerase inhibitor is the far right column. All photos were taken at 200× magnification. Row 1 constituting A, B, and C is hematoxylin and eosin stain. Row 2 consisting of D,E, and F is PSA immunohistochemistry stain. Row 3 consisting of G,H, and I is trichrome stain. BM, bone marrow; Tum, tumor. In A and B control treated mice panels, there is obvious bone destruction and C4-2B cells are seen through out the marrow cavity and invading into the bone (arrow). However, panel C, match treated mouse reveals normal bone marrow and normal calcified osseous bone structures. In panels D and E, on the PSA immunohistochemistry stain and adjacent slices, the brown PSA staining is seen throughout the marrow space and within the bone(arrow), in both the control treated PBS and mismatch telomerase in panels D and E, respectively. However, once again, in panel F, no PSA immunostain is seen in the match treated mouse. Lastly, in panels G and H, adjacent sections, control treated animals reveal bone destruction, whereas match treated animals in panel I reveal a normal architecture. This pattern in these panels was consistent in all the respective mice treatment groups.

Fig. 5

a:Logarithmic conversion of PSA values [ln(1 +PSA)] for Groups I (top), Group II (middle), and Group III (lower) treatment categories, which correspond to control versus single/double versus triple treatment over 16 weeks (see Table I). b:PSA averages for treatment categories Group I, II, and III at weeks 0, 8, 14, and 16. Standard error bars a represent. The triple treatment group had the lowest values, and is seen on the far right. c: PSA averages for the seven treatment groups at weeks 0, 8, and 16, with standard error of the mean (SEM) bars present.

Fig. 6

Example of mice femur H&E staining used for tumor and osteoid measurement in Osteomeasure^™ system. A and C are at 1× magnification, B and D are at 4× magnification. A and B are femurs without tumor, C and D are femurs filled with C4-2B tumor. E and F are at 20 × magnification enlargements of normal and tumor bearing area that circled in B and D. The area used to perform the measurement is boxed. Tum: tumor (open arrow in D), OT: osteoid (open arrow in F), OB: osteoblast (arrow in F), BM: bone marrow, GP: growth plate, WB: newly formed woven bone (arrow in D).