[0085] In this study, cancer stem cells (also referred to as the side population (SP)) and the ATP Binding Cassette (ABC) transporter defining the SP were identified in the following prostate cancer cell lines: DU145wt (parental), DU145/ Pac200 (paclitaxel resistant), and DU145/ Doc50 (docetaxel resistant).

[0086] All examined prostate cancer cell lines: DU145wt, DU145/ Pac200, DU145/ Doc50 exhibit a side population (Figures 1, 3, 5). Importantly, it was found that DU145/ Pac200 and DU145/ Doc50 cells have a very large side population (Figures 1, 5) of about 40 to 60% of their whole cell population consistent with our hypothesis that drug resistant cells can arise from cancer stem cells.

[0087] Taxane resistant cell lines DU145/Pac200 (highly pacitaxel resistant) and DU145/Doc50 (highly docetaxel resistant) together with the parental cell line DU145 as well as the androgen resistant cancer cell lines (LNCaP/C81 and LAPC-4/CSS 100) and their corresponding hormone responsive parental cell lines (LNCaP and LAPC-4) were tested for their response to sodium meta arsenite. Growth curves from standard MTT assays showed that those cell lines respond with similar sensitivity to sodium meta arsenite regardless of their drug resistance (Table 1).

[0088] Each of the prostate cell lines was analyzed for the existence of a cancer stemlike cell fraction using DCV side population analysis. Side populations (SP) based on Dye Cycle Violet (DCV) dye efflux were identified in the drug resistant cell lines, confirming previous results using Hoechst 33342 dye for DU145/Doc50 (not shown). The occurrence of a SP is based on the expression of drug efflux pumps such as P-glycoprotein or BCRP, which are responsible for preventing standard cytotoxic therapy from working. The DU145 prostate cancer cell lines resistant against the standard chemotherapeutic agents paclitaxel and docetaxel had the highest SPs (Table 1). While the hormone resistant cell line LnCaP/C81 and its parental line also showed a clear side population that was suppressible with the drug efflux pump inhibitors verapamil (P-gP inhibitor) or fumitremorgin C (FTC, BCRP inhibitor), a definitive side population for LAPC-4 and its hormone resistant derivate were not detected by this method. Because the hormone therapies, e.g., androgen synthesis inhibitors, are not substrates of drug efflux pumps, these findings are not surprising. Table 1.Percentage of DCV side population in prostate cancer cell lines and in cells pre-treated with the PgP and BRCP transporter inhibitors (Verapamil and Fumitremorgin C, FTC), sensitivity to KML001, telomere length (TRF) and relative telomerase activity (TA).Cell lineSP %SP% with VerapamilSP% with FTCIC50 KML001 [？M]TRF Length [kbp]TA ratioDU1451.040.160.0642.71DU145/Pac200550.0756.5631.05DU145Doc5040.31.3518.3553.40.97LnCaP0.660.120.04431.18LnCaP/C810.160.070.1112.61.10LAPC-41.541.632.2446.71.27LAPC-4/CSS1000.360.190.3034.91.26

[0089] In this study, it was determined that the highly paclitaxel and docetaxel resistant DU145/ Pac200 and DU145/ Doc50 lines have similar telomerase activity and telomere length compared to their parental, drug sensitive DU145 cells (Tab. 1). Table 1.IC50 Values for Taxanes and Telomere Length in Human Prostate Cancer Cell LinesPaclitaxelDocetaxelTAMean TRFNoCell LinesIC50 (？M)Resistance (fold)IC50 (？M)Resistance (fold)Ratio(kb)1DU145 wt0.002510.0005112.52DU145/Pac10N/AN/AN/AN/A0.793.03DU145/Pac2000.351400.24000.962.84Du145 Doc10N/AN/AN/AN/A1.12.25Du145 Doc501400120000.732.8TA = Telomerase activityTRF = Telomere Restriction Fragment

[0090] The SPs of Pac and Doc resistant DU145 cells were inhibited by verapamil, but not FTC, suggesting the Pgp (P-glycoprotein) is the predominant ABC transporter responsible for the occurrence of the SP (Fig. 1, 5). This was consistent with a high surface expression of Pgp by these cells as well as the prostate cancer specific stem cell marker CD44 ((Fig. 1B).

[0091] In this study, the effects of the telomerase inhibitor KML001 (sodium meta arsenite) on DU145wt, DU145/ Pac200, and DU145/ Doc50 cells were compared with that of GRN163L, a telomerase inhibitor that is currently undergoing advanced clinical trials.

[0092] MTT assays of DU145wt, DU145/Doc50 and DU145/ Pac200 treated with KML001 or GRN163L revealed marked inhibition of growth (Tab. 1, Fig. 1, 4). KML001 was equally effective in drug-resistant and parental DU145 cells (Tab. 1, Fig. ID)

[0093] In this study, the ability of sodium meta arsenite and GRN163L to eradicate the side population of DU145wt and DU145/ Pac200 when pre-treated at their IC100 for 48-72 hours were investigated.

[0094] This study demonstrated that DU145 and DU145/Pac200 cell pretreated with KML001 at a drug concentration that inhibits the growth of these cells to 100%, were capable of drastically reducing the SP (Fig. 3B, 5D). However, the mature cells (non-SP) were also eradicated. The specific (telomerase only) inhibitor GRN163L was used to validate effects of sodium meta arsenite on the SP. GRN 163 L also markedly reduced the SP, but was not able to kill non-SP cells to the same extent as KML001 (Fig. 5).

[0095] Overall, the data demonstrate that sodium meta arsenite is effective in eradicating both mature drug-resistant cancer cells and drug-resistant cancer stem cells.

[0096] All prostate cancer cell lines, taxane-resistant and androgen resistant cancer cell lines and hormone responsive parental cells respond with similar sensitivity to KML001 (IC50's of 1-6 ？M). The cancer cell lines that were resistant to paclitaxel and docetaxel were tested using a well-established tumor clonigenic stem cell assay for their response to KML001. (Figure 6) The assay allows only the cancer stem cell population to grow in a soft agar matrix owing to their self-renewal capability. Thus, this assay specifically tests the sensitivity of the stem cell population to an anti-cancer agent.

[0097] The plating efficiency of the cells (2% for DU145 and DU145/Pac200) was similar to the previously reported stem cell population determined in the side population assay (1 % for DU145 and 50% for DU145/Pac200). Similar sensitivity to KML001 for the parental as well as paclitaxel-resistant cell line was observed, as shown in a reduction of colony forming capability in the presence of KML001. A similar IC50 (6 ？M) for the cell lines in this assay was derived as for a standard MTT assay previously conducted (4？M for DU145 and 6？M for DU145/Pac200).

[0098] Taxane resistant cell lines had the highest SPs, while the hormone responsive cell line LAPC-4 and its hormone resistant derivative showed a less definitive SP that was suppressible with the drug efflux inhibitors, verapamil (Pgp inhibitor) and fumitremorgin c (FTC, BCRP inhibitor). The SP positive cell line, DU145, was tested for the presence of CD44 and CD133 and the results showed 0.46% had both markers (data not shown).

[0099] The SP was separated from the bulk tumor mass and growth curves from a standard MTT assay showed that both fractions, the cancer stem cell-like SP positive (SP+) and negative (SP-) fraction, respond with similar sensitivity to KML001. (Figure 7)

[0100] As shown above (Example 3), the cancer stem cell-like fraction identified with the SP assay exhibited higher telomerase activity than the bulk cell fraction. Preliminary studies indicate that the mRNA transcript level of the catalytic subunit of the human telomerase (hTERT) is similar for both populations. (Figure 8A), which may indicate that the higher telomerase activity of the cancer stem cell-like population may be a result of stimulating signaling pathways by telomerase-associated proteins. Preliminary results in the unsorted DU145/Pac200 cell line indicate that KML001 treatment significantly decreases hTERT transcript level at the IC50 and IC100 after 72 hours. (Figure 8B)

[0101] The drug efflux pump (Pgp) is functionally expressed (at the membrane) in the majority of cells in the taxane-resistant prostate cancer cell lines, which is expected since the SP of those cell lines was suppressible with the drug efflux pump inhibitor verapamil. Treatment with KML001 at IC50 for 72 hours reduced the Pgp positive cell population, which corroborates the results of the clonogenic and SP assay.

[0102] The effect of cisplatin in the paclitaxel resistant prostate cancer cell line DU145/Pac200 as well as the parental cell line DU145 was examined in a standard MTT. The results show that both cell lines respond with similar sensitivity (IC50 of 3 and 4 ？M, respectively). Preliminary results indicate a synergistic effect between cisplatin and KML001 in the parental as well as paclitaxel resistant cell lines (results not shown).