Pharmacodynamics of telomerase inhibition and telomere shortening by noncytotoxic suramin.

We reported that suramin is an effective chemosensitizer at noncytotoxic concentrations (<50 µM); this effect was observed in multiple types of human xenograft tumors in vitro and in vivo. Clinical evaluation of noncytotoxic suramin is ongoing. Because (a) suramin inhibits reverse transcriptase, (b) telomerase is a reverse transcriptase, and (c) inhibition of telomerase enhances tumor chemosensitivity, we studied the pharmacodynamics of noncytotoxic suramin on telomerase activity and telomere length in cultured cells and tumors grown in animals. In three human cancer cells that depend on telomerase for telomere maintenance (pharynx FaDu, prostate PC3, breast MCF7), suramin inhibited telomerase activity in cell extracts and intact cells at concentrations that exhibited no cytotoxicity (IC50 of telomerase was between 1 and 3 µM vs. >60 µM for cytotoxicity), and continuous treatment at 10-25 µM for 6 weeks resulted in gradual telomere shortening (maximum of 30%) and cell senescence (measured by ß-galactosidase activity and elevation of mRNA levels of two senescence markers p16 and p21). In contrast, noncytotoxic suramin did not shorten the telomere in telomerase-independent human osteosarcoma Saos-2 cells. In mice bearing FaDu tumors, treatment with noncytotoxic suramin for 6 weeks resulted in telomere erosion in >95% of the tumor cells with an average telomere shortening of >40%. These results indicate noncytotoxic suramin inhibits telomerase, shortens telomere and induces cell senescence, and suggest telomerase inhibition as a potential mechanism of its chemosensitization.

Biodegradable intraprostatic doxorubicin implants.

Systemic chemotherapy is not effective in the treatment of prostate-confined cancer. We developed biodegradable, doxorubicin-loaded cylinders for intraprostatic implantation and evaluated the feasibility of using regional intraprostatic drug therapy to treat prostate-confined cancer. Cylinders were prepared using poly(lactide-co-glycolide) (PLG) or PLG copolymers. The in vitro and in vivo drug release, intraprostatic pharmacokinetics, and histopathology in dogs implanted with the cylinders were studied. The doxorubicin-loaded cylinders made of PLG polymers of 7.9 to 54 kDa molecular weight (MW) had a diameter of ~800 mum, drug loading of 10% to 30% (wt/wt), and even distribution of crystalline drug throughout the matrix. Burst release varied from 3% to 73%, and 7-day cumulative release from 4% to 90%. Decreasing polymer MW and increasing drug loading were associated with higher initial burst release and overall release rates. The in vivo drug release from cylinders (33-kDa PLG, 30% drug loading) in dog prostates was rapid (approximately 80% in 48 hours). Spatial drug distribution, visualized using confocal fluorescence microscopy, showed high concentrations confined to the lobule containing the implant (referred to as the implanted lobule), with steep concentration gradients over the septa separating the lobules. Concentrations in the implanted lobule were about 8 times higher than concentrations delivered by an intravenous injection. The implants caused necrotic cell death in the implanted lobule, without damage to prostatic nerve bundles or the urethra. These results indicate the feasibility of using biodegradable PLG cylinders as intraprostatic implants to selectively deliver high drug concentrations to prostate tissue.

Expression of basic fibroblast growth factor correlates with resistance to paclitaxel in human patient tumors.

BACKGROUND: Preclinical results indicate acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) present in solid tumors as a cause of broad-spectrum chemoresistance, whereas earlier clinical studies suggest that bFGF expression is associated with opposing outcomes in patients. We investigated the relationship between FGF expression and paclitaxel activity in tumors from bladder, breast, head and neck, ovarian, and prostate cancer patients. MATERIALS AND METHODS: Tumors (n = 96) were maintained in three-dimensional histocultures, retaining tumor-stromal interaction. Bladder tumors were treated with paclitaxel for 2 h, and the other tumors for 24 h. Antiproliferative and proapoptotic effects of paclitaxel were quantified and correlated with expression of aFGF, bFGF, P-glycoprotein (Pgp), p53, and bcl-2. RESULTS: Fifty-one percent (49/96) and 63% (61/96) of tumors showed aFGF and bFGF staining, respectively. aFGF expression was positively correlated with tumor stage (p < 0.01), and bFGF expression with tumor grade and Pgp expression (p < 0.05). Paclitaxel inhibited antiproliferation in 86% of tumors (83/96), with an average inhibition of 46 +/- 19% (mean +/- SD) in the responding tumors. Paclitaxel also induced apoptosis in 96% of tumors (92/96), with an average apoptotic index of 12 +/- 7% in the responding tumors. aFGF expression did not correlate with tumor sensitivity to paclitaxel, whereas bFGF expression showed an inverse correlation (p < 0.01). bFGF expression was a stronger predictor of paclitaxel resistance compared to Pgp, p53, or Bcl-2. CONCLUSION: These results support a role of bFGF in paclitaxel resistance in human patient tumors.

Intraprostatic chemotherapy: distribution and transport mechanisms.

PURPOSE: The present study evaluated the tissue distribution and targeting advantage of intraprostatic chemotherapy. EXPERIMENTAL DESIGN: We studied the delivery and spatial distribution of a fluorescent drug, doxorubicin, in the prostate of beagle dogs, after intraprostatic or i.v. administration. Drug concentrations were measured using high-performance liquid chromatography and confocal fluorescence microscopy. RESULTS: I.v. and intraprostatic injections yielded qualitatively and quantitatively different doxorubicin distribution in the prostate. A relatively homogeneous distribution was found after i.v. administration, whereas intraprostatic injection yielded a highly heterogeneous distribution with >10-fold higher concentrations localized in a cone-shaped glandular lobule bound by fibromuscular stroma, compared with other parts of the prostate. Compared with i.v. injection, intraprostatic injection yielded, on average, approximately 100-fold higher tissue-to-plasma concentration ratio, ranging from 963-fold near the injection site to 19-fold in the contralateral half of the prostate. The drug distribution within the prostate further suggests an important role for acinar flow in intraprostatic drug transport. CONCLUSIONS: Intraprostatic administration represents a viable option to deliver high drug concentrations within the prostate. The results further suggest the fibromuscular stroma separating the prostatic lobules as a major barrier to drug transport and convective flow as an important drug transport mechanism in the prostate.

Synergy between 3'-azido-3'-deoxythymidine and paclitaxel in human pharynx FaDu cells.

PURPOSE: We recently demonstrated simultaneous targeting of telomere and telomerase as a novel cancer therapeutic approach, and that telomerase inhibitors such as 3'-azido-3'-deoxythymidine (AZT) significantly enhanced the antitumor activity of paclitaxel, which causes telomere erosion, in telomerase-positive human pharynx FaDu tumors in vitro and in vivo. The present study evaluated the synergy between AZT and paclitaxel to identify optimal combinations for future clinical evaluation. METHODS: FaDu cells were incubated with or without AZT for 24 h and then treated with AZT with or without paclitaxel for an additional 48 h. Under these conditions, single agent paclitaxel produced a 60% maximum reduction of cell number (IC50) was 7.3 nM), and single agent AZT produced a 97% reduction (IC50 was 5.6 microM). Synergy was evaluated using fixed-concentration and fixed-ratio methods, and data were analyzed by the combination index method. RESULTS: The results indicate a concentration-dependent synergy between the two drugs; the synergy was higher for combinations containing greater paclitaxel-to-AZT concentration ratios and increased with the level of drug effect. For example, in combinations containing 1 microM AZT, synergy was 1.3-fold at the 20% effect level and 3.1-fold at the 60% effect level. Because the major antitumor activity, determined by comparing the posttreatment cell number to the pretreatment cell number, was antiproliferation at the 20% effect level and cell kill at the 60% effect level, our results suggest that AZT mainly enhances the cell kill effect of paclitaxel. CONCLUSION: In summary, the present study demonstrates a synergistic interaction between paclitaxel and AZT and supports a combination using a low and nontoxic AZT dose in combination with a therapeutically active dose of paclitaxel.

Simultaneous targeting of telomeres and telomerase as a cancer therapeutic approach.

Telomeres, which are important for maintaining chromosome integrity and functions, shorten with each cell division. Telomerase, responsible for telomere synthesis, is expressed in approximately 90% of human tumor cells but seldom in normal somatic cells. This study evaluated the hypothesis that simultaneous shortening of telomeres and inhibition of telomerase results in synergistic and tumor-selective cytotoxicity. In telomerase-positive human pharynx FaDu tumor cells, paclitaxel caused telomere erosion (first detected at 1 h) and apoptosis. Expression of antisense to the RNA component of human telomerase (hTR) inhibited telomerase activity, shortened telomere length, reduced cell growth rate, and resulted in a significant higher sensitivity to paclitaxel. Another telomerase inhibitor, 3'-azido-3'-deoxythymidine (AZT), at a concentration that produced little or no cell detachment or apoptosis, inhibited the telomerase activity and enhanced the paclitaxel-induced cell detachment and apoptosis. AZT also enhanced the activity of paclitaxel in mice bearing well-established s.c. FaDu xenograft tumors (i.e., reduced residual tumor size, enhanced apoptotic cell fraction, and prolonged survival time), without enhancing host toxicity. In contrast, AZT did not enhance the paclitaxel activity in the telomerase-negative osteosarcoma Saos-2 cells nor in FaDu cells where telomerase was already suppressed by antisense hTR, confirming that the AZT effect in parent FaDu cells is mediated through telomerase inhibition. These results demonstrate that combined use of agents targeting both telomere and telomerase yielded synergistic activity selective for tumors that depend on telomerase for telomere maintenance.

Telomere maintenance in telomerase-positive human ovarian SKOV-3 cells cannot be retarded by complete inhibition of telomerase.

The two known mechanisms for telomere maintenance in eukaryocytes are telomerase in telomerase-positive cells and alternative lengthening of telomeres (ALT) in telomerase-negative cells. We report here that telomere maintenance in the telomerase-positive human ovarian SKOV-3 cells was not affected by inhibition of telomerase. For comparison, the effect of telomerase inhibitors on telomere maintenance in another telomerase-positive cell line (i.e. human pharynx FaDu cells) and the telomerase-negative human osteosarcoma Saos-2 cells was examined. Telomerase activity was measured using a modified telomeric repeat amplification protocol and telomere length was measured using a solution hybridization-based method and fluorescence in situ hybridization. A reverse transcriptase inhibitor (3'-azido-deoxythymidine or AZT) and an antisense against a component of human telomerase RNA (antisense hTR) were used to inhibit telomerase. FaDu and SKOV-3 cells showed comparable baseline telomerase activity. Telomerase activity in both cells was inhibited about equally by AZT (maximal inhibition of approximately 80%) and by expression of antisense hTR (complete inhibition in SKOV-3 cells and maximal inhibition of approximately 80% in FaDu cells). However, treatment with telomerase inhibitors resulted in approximately 50% telomere shortening in FaDu cells but had no effect on SKOV-3 nor Saos-2 cells. SKOV-3 cells did not show the characteristic features of ALT (i.e. heterogeneous telomere length and promyelocytic leukemia bodies), whereas these ALT features were observed in Saos-2 cells. Collectively, these results suggest the existence of a telomerase-independent mechanism of telomere maintenance in the telomerase-positive SKOV-3 cells.